Macrocyclic complexes of alpha-emitting radionuclides and their use in targeted radiotherapy of cancer

ABSTRACT

The present technology provides compounds as well as compositions including such compounds useful in targeted radiotherapy of cancer and/or mammalian tissue overexpressing prostate specific membrane antigen (“PSMA”) where the compounds are represented by the following: 
     
       
         
         
             
             
         
       
         
         
           
             or a pharmaceutically acceptable salt thereof, 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             or a pharmaceutically acceptable salt thereof, 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             or a pharmaceutically acceptable salt thereof,
 
wherein M 1  is independently at each occurrence an alpha-emitting radionuclide. Equivalents of such compounds are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.16/689,856, filed Nov. 20, 2019, which claims the benefit of andpriority to U.S. Provisional Patent Application No. 62/769,989, filedNov. 20, 2018, U.S. Provisional Patent Application No. 62/788,700, filedJan. 4, 2019, and U.S. Provisional Patent Application No. 62/792,835,filed Jan. 15, 2019, each of which is incorporated herein by referencein its entirety for any and all purposes.

U.S. GOVERNMENT RIGHTS

This invention was made with government support under UL1TR00457 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD

The present technology generally relates to macrocyclic complexes ofalpha-emitting radionuclides, as well as compositions including suchcompounds and methods of use.

SUMMARY

In an aspect, a compound of Formula I is provided:

or a pharmaceutically acceptable salt thereof, wherein

-   -   Z¹ is H or —X¹—W²;    -   Z² is OH or NH—W³;    -   Z³ is H or W⁷;    -   α is 0 or 1;    -   X is O, NH, or S;    -   W² and W³ are each independently H, alkyl, cycloalkyl, alkenyl,        cycloalkenyl, alkynyl, aryl, heterocyclyl, heteroaryl,        —CH₂CH₂—(OCH₂CH₂)_(w)—R′ where w is 1, 2, 3, 4, 5, 6, 7, 8, 9,        or 10, or —CH₂CH₂—(OCH₂CH₂)_(x)—OR′ where x is 1, 2, 3, 4, 5, 6,        7, 8, 9, or 10, each of which may optionally be substituted with        one or more of halo, —N₃, —OR′, —CH₂CH₂—(OCH₂CH₂)_(x)—R′ where y        is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —CH₂CH₂—(OCH₂CH₂)_(z)—OR′        where z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —SR′, —OC(O)R′,        —C(O)OR′, —C(S)OR′, —S(O)R′, —SO₂R′, —SO₂(OR′), —SO₂NR′₂,        —P(O)(OR′)₂, —P(O)R′(OR′), —P(O)R′₂, —CN, —OCN, —SCN, —NCO,        —NCS, —NR′—NH₂, —N═C═N—R′, —SO₂Cl, —C(O)Cl, or an epoxide group;    -   W⁵ and W⁷ are each independently OH, NH₂, SH, alkyl, cycloalkyl,        alkenyl, cycloalkenyl, alkynyl, aryl, heterocyclyl, heteroaryl,        —CH₂CH₂—(OCH₂CH₂)_(w)—R′ where w is 1, 2, 3, 4, 5, 6, 7, 8, 9,        or 10, or —CH₂CH₂—(OCH₂CH₂)_(x)—OR′ where x is 1, 2, 3, 4, 5, 6,        7, 8, 9, or 10, each of which may optionally be substituted with        one or more of halo, —N₃, —OR′, —CH₂CH₂—(OCH₂CH₂)y_(x)-R′ where        y is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —CH₂CH₂—(OCH₂CH₂)_(z)—OR′        where z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —SR′, —OC(O)R′,        —C(O)OR′, —C(S)OR′, —S(O)R′, —SO₂R′, —SO₂(OR′), —SO₂NR′₂,        —P(O)(OR′)₂, —P(O)R′(OR′), —P(O)R′₂, —CN, —OCN, —SCN, —NCO,        —NCS, —NR′—NH₂, —N═C═N—R′, —SO₂Cl, —C(O)Cl, or an epoxide group;        and    -   R′ is independently at each occurrence H, halo, —N₃, C₁-C₆        alkyl, C₃-C₆ cycloalkyl, C₂-C₆ alkenyl, C₅-C₈ cycloalkenyl,        C₂-C₆ alkynyl, C₈-C₁₀ cycloalkynyl, C₅-C₆ aryl, heterocyclyl, or        heteroaryl.

In a related aspect, a compound of Formula IA is provided

or a pharmaceutically acceptable salt thereof, wherein

-   -   M¹ is an alpha-emitting radionuclide;    -   Z¹ is H or —X¹—W²;    -   Z² is OH or NH—W³;    -   Z³ is H or W;    -   α is 0 or 1;    -   X¹ is O, NH, or S;    -   W² and W³ are each independently H, alkyl, cycloalkyl, alkenyl,        cycloalkenyl, alkynyl, aryl, heterocyclyl, heteroaryl,        —CH₂CH₂—(OCH₂CH₂)_(w)—R′ where w is 1, 2, 3, 4, 5, 6, 7, 8, 9,        or 10, or —CH₂CH₂—(OCH₂CH₂)_(x)—OR′ where x is 1, 2, 3, 4, 5, 6,        7, 8, 9, or 10, each of which may optionally be substituted with        one or more of halo, —N₃, —OR′, —CH₂CH₂—(OCH₂CH₂)_(y)—R′ where y        is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —CH₂CH₂—(OCH₂CH₂)_(z)—OR′        where z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —SR′, —OC(O)R′,        —C(O)OR′, —C(S)OR′, —S(O)R′, —SO₂R′, —SO₂(OR′), —SO₂NR′₂,        —P(O)(OR′)₂, —P(O)R′(OR′), —P(O)R′₂, —CN, —OCN, —SCN, —NCO,        —NCS, —NR′—NH₂, —N═C═N—R′, —SO₂Cl, —C(O)Cl, or an epoxide group;    -   W⁵ and W⁷ are each independently OH, NH₂, SH, alkyl, cycloalkyl,        alkenyl, cycloalkenyl, alkynyl, aryl, heterocyclyl, heteroaryl,        —CH₂CH₂—(OCH₂CH₂)_(w)—R′ where w is 1, 2, 3, 4, 5, 6, 7, 8, 9,        or 10, or —CH₂CH₂—(OCH₂CH₂)_(x)—OR′ where x is 1, 2, 3, 4, 5, 6,        7, 8, 9, or 10, each of which may optionally be substituted with        one or more of halo, —N₃, —OR′, —CH₂CH₂—(OCH₂CH₂)y_(x)-R′ where        y is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —CH₂CH₂—(OCH₂CH₂)_(z)—OR′        where z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —SR′, —OC(O)R′,        —C(O)OR′, —C(S)OR′, —S(O)R′, —SO₂R′, —SO₂(OR′), —SO₂NR′₂,        —P(O)(OR′)₂, —P(O)R′(OR′), —P(O)R′₂, —CN, —OCN, —SCN, —NCO,        —NCS, —NR′—NH₂, —N═C═N—R′, —SO₂Cl, —C(O)Cl, or an epoxide group;        and    -   R′ is independently at each occurrence H, halo, —N₃, C₁-C₆        alkyl, C₃-C₆ cycloalkyl, C₂-C₆ alkenyl, C₅-C₈ cycloalkenyl,        C₂-C₆ alkynyl, C₈-C₁₀ cycloalkynyl, C₅-C₆ aryl, heterocyclyl, or        heteroaryl.

In a further related aspect, the present technology provides a compounduseful in targeted radiotherapy of cancer and/or mammalian tissueoverexpressing prostate specific membrane antigen (“PSMA”) (a “targetingcompound”) where the compound is of Formula II

or a pharmaceutically acceptable salt thereof, wherein

-   -   M¹ is an alpha-emitting radionuclide;    -   Z¹ is H or -L³-R²²;    -   Z² is OH or NH-L⁴-R²⁴;    -   Z³ is H or -L⁶-R²⁸;    -   α is 0 or 1;    -   X¹ is O, NH, or S;    -   L³, L⁴, L⁵, and L⁶ are independently at each occurrence a bond        or a linker group; and    -   R²², R²⁴, R²⁶, and R²⁸ each independently comprises an antibody,        antibody fragment (e.g., an antigen-binding fragment), a binding        moiety, a binding peptide, a binding polypeptide (such as a        selective targeting oligopeptide containing up to 50 amino        acids), a binding protein, an enzyme, a nucleobase-containing        moiety (such as an oligonucleotide, DNA or RNA vector, or        aptamer), or a lectin.

In a further related aspect, a modified antibody, modified antibodyfragment, or modified binding peptide comprising a linkage arising fromconjugation of a compound of Formula I or pharmaceutically acceptablesalt thereof, with an antibody, antibody fragment, or binding peptide.In a related aspect, a modified antibody, modified antibody fragment, ormodified binding peptide is provided that includes a linkage arisingfrom conjugation of a compound of Formula IA or a pharmaceuticallyacceptable salt thereof, with an antibody, antibody fragment, or bindingpeptide.

In any embodiment and/or aspect disclosed herein (for simplicity's sake,hereinafter recited as “in any embodiment disclosed herein” or thelike), it may be that the antibody includes belimumab, Mogamulizumab,Blinatumomab, Ibritumomab tiuxetan, Obinutuzumab, Ofatumumab, Rituximab,Inotuzumab ozogamicin, Moxetumomab pasudotox, Brentuximab vedotin,Daratumumab, Ipilimumab, Cetuximab, Necitumumab, Panitumumab,Dinutuximab, Pertuzumab, Trastuzumab, Trastuzumab emtansine, Siltuximab,Cemiplimab, Nivolumab, Pembrolizumab, Olaratumab, Atezolizumab,Avelumab, Durvalumab, Capromab pendetide, Elotuzumab, Denosumab,Ziv-aflibercept, Bevacizumab, Ramucirumab, Tositumomab, Gemtuzumabozogamicin, Alemtuzumab, Cixutumumab, Girentuximab, Nimotuzumab,Catumaxomab, or Etaracizumab. In any embodiment disclosed herein, it maybe that the antibody fragment includes an antigen-binding fragment ofbelimumab, Mogamulizumab, Blinatumomab, Ibritumomab tiuxetan,Obinutuzumab, Ofatumumab, Rituximab, Inotuzumab ozogamicin, Moxetumomabpasudotox, Brentuximab vedotin, Daratumumab, Ipilimumab, Cetuximab,Necitumumab, Panitumumab, Dinutuximab, Pertuzumab, Trastuzumab,Trastuzumab emtansine, Siltuximab, Cemiplimab, Nivolumab, Pembrolizumab,Olaratumab, Atezolizumab, Avelumab, Durvalumab, Capromab pendetide,Elotuzumab, Denosumab, Ziv-aflibercept, Bevacizumab, Ramucirumab,Tositumomab, Gemtuzumab ozogamicin, Alemtuzumab, Cixutumumab,Girentuximab, Nimotuzumab, Catumaxomab, or Etaracizumab. In anyembodiment disclosed herein, it may be that the binding peptide includesa prostate specific membrane antigen (“PSMA”) binding peptide, asomatostatin receptor agonist, a bombesin receptor agonist, a seprasebinding compound, or a binding fragment thereof.

In another aspect, the present technology also provides compositions(e.g., pharmaceutical compositions) and medicaments comprising any ofone of the embodiments of the compounds of Formulas I, IA, or II (or apharmaceutically acceptable salt thereof) disclosed herein and apharmaceutically acceptable carrier or one or more excipients orfillers. In a similar aspect, the present technology also providescompositions (e.g., pharmaceutical compositions) and medicamentscomprising any of one of the embodiments of the modified antibody,modified antibody fragment, or modified binding peptide of the presenttechnology disclosed herein and a pharmaceutically acceptable carrier orone or more excipients or fillers.

In an aspect, a method of treating a subject is provided, wherein themethod includes administering a targeting compound of the presenttechnology to the subject or administering a modified antibody, modifiedantibody fragment, or modified binding peptide of the present technologyto the subject. In any embodiment disclosed herein, it may be that thesubject suffers from cancer and/or mammalian tissue overexpressingprostate specific membrane antigen (“PSMA”).

In an aspect, a compound is provided that includes a first domain havinga blood-protein binding moiety with low specific affinity for theblood-protein, a second domain having a tumor targeting moiety with highaffinity for a tumor antigen, and a third domain having a chelator.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B shows x-ray crystal structures of[La(Hmacropa)(H₂O)].(ClO₄)₂ (FIG. 1A, side view; FIG. 2B, top view).FIGS. 1C and 1D shows x-ray crystal structures of [Lu(macropa)].ClO₄.DMF(FIG. 1C, side view; FIG. 1D, top view). Ellipsoids are drawn at the 50%probability level. Counteranions and hydrogen atoms attached to carbonsare omitted for clarity.

FIGS. 2A-C shows the biodistribution of ²²⁵Ac(NO₃)₃ (FIG. 2A),[²²⁵Ac(macropa)]⁺ (FIG. 2B), and [²²⁵Ac(DOTA)]⁻ (FIG. 2C) for selectorgans following intravenous injection in mice. Adult C57BL/6 mice weresacrificed 15 min, 1 h, or 5 h post injection. Values for each timepoint are given as mean % ID/g±1 SD.

FIG. 3 provides a schematic overview of the synthesis ofMacropa-(OCH₂CH₂)-Ph-NCS (an embodiment of the present technology).

DETAILED DESCRIPTION

The following terms are used throughout as defined below.

As used herein and in the appended claims, singular articles such as “a”and “an” and “the” and similar referents in the context of describingthe elements (especially in the context of the following claims) are tobe construed to cover both the singular and the plural, unless otherwiseindicated herein or clearly contradicted by context. Recitation ofranges of values herein are merely intended to serve as a shorthandmethod of referring individually to each separate value falling withinthe range, unless otherwise indicated herein, and each separate value isincorporated into the specification as if it were individually recitedherein. All methods described herein can be performed in any suitableorder unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein, is intended merely to betterilluminate the embodiments and does not pose a limitation on the scopeof the claims unless otherwise stated. No language in the specificationshould be construed as indicating any non-claimed element as essential.

As used herein, “about” will be understood by persons of ordinary skillin the art and will vary to some extent depending upon the context inwhich it is used. If there are uses of the term which are not clear topersons of ordinary skill in the art, given the context in which it isused, “about” will mean up to plus or minus 10% of the particularterm—for example, “about 10 wt. %” would be understood to mean “9 wt. %to 11 wt. %.” It is to be understood that when “about” precedes a term,the term is to be construed as disclosing “about” the term as well asthe term without modification by “about”—for example, “about 10 wt. %”discloses “9 wt. % to 11 wt. %” as well as disclosing “10 wt. %.”

Generally, reference to a certain element such as hydrogen or H is meantto include all isotopes of that element. For example, if an R group isdefined to include hydrogen or H, it also includes deuterium andtritium. Compounds comprising radioisotopes such as tritium, C¹⁴, P³²and S³⁵ are thus within the scope of the present technology. Proceduresfor inserting such labels into the compounds of the present technologywill be readily apparent to those skilled in the art based on thedisclosure herein.

In general, “substituted” refers to an organic group as defined below(e.g., an alkyl group) in which one or more bonds to a hydrogen atomcontained therein are replaced by a bond to non-hydrogen or non-carbonatoms. Substituted groups also include groups in which one or more bondsto a carbon(s) or hydrogen(s) atom are replaced by one or more bonds,including double or triple bonds, to a heteroatom. Thus, a substitutedgroup is substituted with one or more substituents, unless otherwisespecified. In some embodiments, a substituted group is substituted with1, 2, 3, 4, 5, or 6 substituents. Examples of substituent groupsinclude: halogens (i.e., F, Cl, Br, and I); hydroxyls; alkoxy, alkenoxy,aryloxy, aralkyloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy,and heterocyclylalkoxy groups; carbonyls (oxo); carboxylates; esters;urethanes; oximes; hydroxylamines; alkoxyamines; aralkoxyamines; thiols;sulfides; sulfoxides; sulfones; sulfonyls; pentafluorosulfanyl (i.e.,SF₅), sulfonamides; amines; N-oxides; hydrazines; hydrazides;hydrazones; azides; amides; ureas; amidines; guanidines; enamines;imides; isocyanates; isothiocyanates; cyanates; thiocyanates; imines;nitro groups; nitriles (i.e., CN); and the like.

Substituted ring groups such as substituted cycloalkyl, aryl,heterocyclyl and heteroaryl groups also include rings and ring systemsin which a bond to a hydrogen atom is replaced with a bond to a carbonatom. Therefore, substituted cycloalkyl, aryl, heterocyclyl andheteroaryl groups may also be substituted with substituted orunsubstituted alkyl, alkenyl, and alkynyl groups as defined below.

As used herein, C_(m)-C_(n), such as C₁-C₁₂, C₁-C₈, or C₁-C₆ when usedbefore a group refers to that group containing m to n carbon atoms.

Alkyl groups include straight chain and branched chain alkyl groupshaving from 1 to 12 carbon atoms, and typically from 1 to 10 carbons or,in some embodiments, from 1 to 8, 1 to 6, or 1 to 4 carbon atoms.Examples of straight chain alkyl groups include groups such as methyl,ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octylgroups. Examples of branched alkyl groups include, but are not limitedto, isopropyl, iso-butyl, sec-butyl, tert-butyl, neopentyl, isopentyl,and 2,2-dimethylpropyl groups. Alkyl groups may be substituted orunsubstituted. Representative substituted alkyl groups may besubstituted one or more times with substituents such as those listedabove, and include without limitation haloalkyl (e.g., trifluoromethyl),hydroxyalkyl, thioalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl,alkoxyalkyl, carboxyalkyl, and the like.

Cycloalkyl groups include mono-, bi- or tricyclic alkyl groups havingfrom 3 to 12 carbon atoms in the ring(s), or, in some embodiments, 3 to10, 3 to 8, or 3 to 4, 5, or 6 carbon atoms. Exemplary monocycliccycloalkyl groups include, but not limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In someembodiments, the cycloalkyl group has 3 to 8 ring members, whereas inother embodiments the number of ring carbon atoms range from 3 to 5, 3to 6, or 3 to 7. Bi- and tricyclic ring systems include both bridgedcycloalkyl groups and fused rings, such as, but not limited to,bicyclo[2.1.1]hexane, adamantyl, decalinyl, and the like. Cycloalkylgroups may be substituted or unsubstituted. Substituted cycloalkylgroups may be substituted one or more times with, non-hydrogen andnon-carbon groups as defined above. However, substituted cycloalkylgroups also include rings that are substituted with straight or branchedchain alkyl groups as defined above. Representative substitutedcycloalkyl groups may be mono-substituted or substituted more than once,such as, but not limited to, 2,2-, 2,3-, 2,4-2,5- or 2,6-disubstitutedcyclohexyl groups, which may be substituted with substituents such asthose listed above.

Cycloalkylalkyl groups are alkyl groups as defined above in which ahydrogen or carbon bond of an alkyl group is replaced with a bond to acycloalkyl group as defined above. In some embodiments, cycloalkylalkylgroups have from 4 to 16 carbon atoms, 4 to 12 carbon atoms, andtypically 4 to 10 carbon atoms. Cycloalkylalkyl groups may besubstituted or unsubstituted. Substituted cycloalkylalkyl groups may besubstituted at the alkyl, the cycloalkyl or both the alkyl andcycloalkyl portions of the group. Representative substitutedcycloalkylalkyl groups may be mono-substituted or substituted more thanonce, such as, but not limited to, mono-, di- or tri-substituted withsubstituents such as those listed above.

Alkenyl groups include straight and branched chain alkyl groups asdefined above, except that at least one double bond exists between twocarbon atoms. Alkenyl groups have from 2 to 12 carbon atoms, andtypically from 2 to 10 carbons or, in some embodiments, from 2 to 8, 2to 6, or 2 to 4 carbon atoms. In some embodiments, the alkenyl group hasone, two, or three carbon-carbon double bonds. Examples include, but arenot limited to vinyl, allyl, —CH═CH(CH₃), —CH═C(CH₃)₂, —C(CH₃)═CH₂,—C(CH₃)═CH(CH₃), —C(CH₂CH₃)═CH₂, among others. Alkenyl groups may besubstituted or unsubstituted. Representative substituted alkenyl groupsmay be mono-substituted or substituted more than once, such as, but notlimited to, mono-, di- or tri-substituted with substituents such asthose listed above.

Cycloalkenyl groups include cycloalkyl groups as defined above, havingat least one double bond between two carbon atoms. Cycloalkenyl groupsmay be substituted or unsubstituted. In some embodiments thecycloalkenyl group may have one, two or three double bonds but does notinclude aromatic compounds. Cycloalkenyl groups have from 4 to 14 carbonatoms, or, in some embodiments, 5 to 14 carbon atoms, 5 to 10 carbonatoms, or even 5, 6, 7, or 8 carbon atoms. Examples of cycloalkenylgroups include cyclohexenyl, cyclopentenyl, cyclohexadienyl,cyclobutadienyl, and cyclopentadienyl.

Cycloalkenylalkyl groups are alkyl groups as defined above in which ahydrogen or carbon bond of the alkyl group is replaced with a bond to acycloalkenyl group as defined above. Cycloalkenylalkyl groups may besubstituted or unsubstituted. Substituted cycloalkenylalkyl groups maybe substituted at the alkyl, the cycloalkenyl or both the alkyl andcycloalkenyl portions of the group. Representative substitutedcycloalkenylalkyl groups may be substituted one or more times withsubstituents such as those listed above.

Alkynyl groups include straight and branched chain alkyl groups asdefined above, except that at least one triple bond exists between twocarbon atoms. Alkynyl groups have from 2 to 12 carbon atoms, andtypically from 2 to 10 carbons or, in some embodiments, from 2 to 8, 2to 6, or 2 to 4 carbon atoms. In some embodiments, the alkynyl group hasone, two, or three carbon-carbon triple bonds. Examples include, but arenot limited to —C≡CH, —C≡CCH₃, —CH₂C≡CCH₃, —C≡CCH₂CH(CH₂CH)₂, amongothers. Alkynyl groups may be substituted or unsubstituted.Representative substituted alkynyl groups may be mono-substituted orsubstituted more than once, such as, but not limited to, mono-, di- ortri-substituted with substituents such as those listed above.

Aryl groups are cyclic aromatic hydrocarbons that do not containheteroatoms. Aryl groups herein include monocyclic, bicyclic andtricyclic ring systems. Thus, aryl groups include, but are not limitedto, phenyl, azulenyl, heptalenyl, biphenyl, fluorenyl, phenanthrenyl,anthracenyl, indenyl, indanyl, pentalenyl, and naphthyl groups. In someembodiments, aryl groups contain 6-14 carbons, and in others from 6 to12 or even 6-10 carbon atoms in the ring portions of the groups. In someembodiments, the aryl groups are phenyl or naphthyl. Aryl groups may besubstituted or unsubstituted. The phrase “aryl groups” includes groupscontaining fused rings, such as fused aromatic-aliphatic ring systems(e.g., indanyl, tetrahydronaphthyl, and the like). Representativesubstituted aryl groups may be mono-substituted or substituted more thanonce. For example, monosubstituted aryl groups include, but are notlimited to, 2-, 3-, 4-, 5-, or 6-substituted phenyl or naphthyl groups,which may be substituted with substituents such as those listed above.

Aralkyl groups are alkyl groups as defined above in which a hydrogen orcarbon bond of an alkyl group is replaced with a bond to an aryl groupas defined above. In some embodiments, aralkyl groups contain 7 to 16carbon atoms, 7 to 14 carbon atoms, or 7 to 10 carbon atoms. Aralkylgroups may be substituted or unsubstituted. Substituted aralkyl groupsmay be substituted at the alkyl, the aryl or both the alkyl and arylportions of the group. Representative aralkyl groups include but are notlimited to benzyl and phenethyl groups and fused (cycloalkylaryl)alkylgroups such as 4-indanylethyl. Representative substituted aralkyl groupsmay be substituted one or more times with substituents such as thoselisted above.

Heterocyclyl groups include aromatic (also referred to as heteroaryl)and non-aromatic ring compounds containing 3 or more ring members, ofwhich one or more is a heteroatom such as, but not limited to, N, O, andS. In some embodiments, the heterocyclyl group contains 1, 2, 3 or 4heteroatoms. In some embodiments, heterocyclyl groups include mono-, bi-and tricyclic rings having 3 to 16 ring members, whereas other suchgroups have 3 to 6, 3 to 10, 3 to 12, or 3 to 14 ring members.Heterocyclyl groups encompass aromatic, partially unsaturated andsaturated ring systems, such as, for example, imidazolyl, imidazolinyland imidazolidinyl groups. The phrase “heterocyclyl group” includesfused ring species including those comprising fused aromatic andnon-aromatic groups, such as, for example, benzotriazolyl,2,3-dihydrobenzo[1,4]dioxinyl, and benzo[1,3]dioxolyl. The phrase alsoincludes bridged polycyclic ring systems containing a heteroatom suchas, but not limited to, quinuclidyl. Heterocyclyl groups may besubstituted or unsubstituted. Heterocyclyl groups include, but are notlimited to, aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl,pyrazolidinyl, thiazolidinyl, tetrahydrothiophenyl, tetrahydrofuranyl,dioxolyl, furanyl, thiophenyl, pyrrolyl, pyrrolinyl, imidazolyl,imidazolinyl, pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl, oxazolyl,isoxazolyl, thiazolyl, thiazolinyl, isothiazolyl, thiadiazolyl,oxadiazolyl, piperidyl, piperazinyl, morpholinyl, thiomorpholinyl,tetrahydropyranyl, tetrahydrothiopyranyl, oxathiane, dioxyl, dithianyl,pyranyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl,dihydropyridyl, dihydrodithiinyl, dihydrodithionyl, homopiperazinyl,quinuclidyl, indolyl, indolinyl, isoindolyl, azaindolyl(pyrrolopyridyl), indazolyl, indolizinyl, benzotriazolyl,benzimidazolyl, benzofuranyl, benzothiophenyl, benzthiazolyl,benzoxadiazolyl, benzoxazinyl, benzodithiinyl, benzoxathiinyl,benzothiazinyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl,benzo[1,3]dioxolyl, pyrazolopyridyl, imidazopyridyl (azabenzimidazolyl),triazolopyridyl, isoxazolopyridyl, purinyl, xanthinyl, adeninyl,guaninyl, quinolinyl, isoquinolinyl, quinolizinyl, quinoxalinyl,quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl, pteridinyl,thianaphthyl, dihydrobenzothiazinyl, dihydrobenzofuranyl,dihydroindolyl, dihydrobenzodioxinyl, tetrahydroindolyl,tetrahydroindazolyl, tetrahydrobenzimidazolyl, tetrahydrobenzotriazolyl,tetrahydropyrrolopyridyl, tetrahydropyrazolopyridyl,tetrahydroimidazopyridyl, tetrahydrotriazolopyridyl, andtetrahydroquinolinyl groups. Representative substituted heterocyclylgroups may be mono-substituted or substituted more than once, such as,but not limited to, pyridyl or morpholinyl groups, which are 2-, 3-, 4-,5-, or 6-substituted, or disubstituted with various substituents such asthose listed above.

Heteroaryl groups are aromatic ring compounds containing 5 or more ringmembers, of which, one or more is a heteroatom such as, but not limitedto, N, O, and S. Heteroaryl groups include, but are not limited to,groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl,isoxazolyl, thiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl,thiophenyl, benzothiophenyl, furanyl, benzofuranyl, indolyl, azaindolyl(pyrrolopyridinyl), indazolyl, benzimidazolyl, imidazopyridinyl(azabenzimidazolyl), pyrazolopyridinyl, triazolopyridinyl,benzotriazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl,imidazopyridinyl, isoxazolopyridinyl, thianaphthyl, purinyl, xanthinyl,adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl,quinoxalinyl, and quinazolinyl groups. Heteroaryl groups include fusedring compounds in which all rings are aromatic such as indolyl groupsand include fused ring compounds in which only one of the rings isaromatic, such as 2,3-dihydro indolyl groups. Heteroaryl groups may besubstituted or unsubstituted. Thus, the phrase “heteroaryl groups”includes fused ring compounds as well as includes heteroaryl groups thathave other groups bonded to one of the ring members, such as alkylgroups. Representative substituted heteroaryl groups may be substitutedone or more times with various substituents such as those listed above.

Heterocyclylalkyl groups are alkyl groups as defined above in which ahydrogen or carbon bond of an alkyl group is replaced with a bond to aheterocyclyl group as defined above. Heterocyclylalkyl groups may besubstituted or unsubstituted. Substituted heterocyclylalkyl groups maybe substituted at the alkyl, the heterocyclyl or both the alkyl andheterocyclyl portions of the group. Representative heterocyclyl alkylgroups include, but are not limited to, morpholin-4-yl-ethyl,furan-2-yl-methyl, imidazol-4-yl-methyl, pyridin-3-yl-methyl,tetrahydrofuran-2-yl-ethyl, and indol-2-yl-propyl. Representativesubstituted heterocyclylalkyl groups may be substituted one or moretimes with substituents such as those listed above.

Heteroaralkyl groups are alkyl groups as defined above in which ahydrogen or carbon bond of an alkyl group is replaced with a bond to aheteroaryl group as defined above. Heteroaralkyl groups may besubstituted or unsubstituted. Substituted heteroaralkyl groups may besubstituted at the alkyl, the heteroaryl or both the alkyl andheteroaryl portions of the group. Representative substitutedheteroaralkyl groups may be substituted one or more times withsubstituents such as those listed above.

Groups described herein having two or more points of attachment (i.e.,divalent, trivalent, or polyvalent) within the compound of the presenttechnology are designated by use of the suffix, “ene.” For example,divalent alkyl groups are alkylene groups, divalent aryl groups arearylene groups, divalent heteroaryl groups are divalent heteroarylenegroups, and so forth. Substituted groups having a single point ofattachment to the compound of the present technology are not referred tousing the “ene” designation. Thus, e.g., chloroethyl is not referred toherein as chloroethylene. Such groups may further be substituted orunsubstituted.

Alkoxy groups are hydroxyl groups (—OH) in which the bond to thehydrogen atom is replaced by a bond to a carbon atom of a substituted orunsubstituted alkyl group as defined above. Examples of linear alkoxygroups include but are not limited to methoxy, ethoxy, propoxy, butoxy,pentoxy, hexoxy, and the like. Examples of branched alkoxy groupsinclude but are not limited to isopropoxy, sec-butoxy, tert-butoxy,isopentoxy, isohexoxy, and the like. Examples of cycloalkoxy groupsinclude but are not limited to cyclopropyloxy, cyclobutyloxy,cyclopentyloxy, cyclohexyloxy, and the like. Alkoxy groups may besubstituted or unsubstituted. Representative substituted alkoxy groupsmay be substituted one or more times with substituents such as thoselisted above.

The terms “alkanoyl” and “alkanoyloxy” as used herein can refer,respectively, to —C(O)-alkyl and —O—C(O)-alkyl groups, where in someembodiments the alkanoyl or alkanoyloxy groups each contain 2-5 carbonatoms. Similarly, the terms “aryloyl” and “aryloyloxy” respectivelyrefer to —C(O)-aryl and —O—C(O)-aryl groups.

The terms “aryloxy” and “arylalkoxy” refer to, respectively, asubstituted or unsubstituted aryl group bonded to an oxygen atom and asubstituted or unsubstituted aralkyl group bonded to the oxygen atom atthe alkyl. Examples include but are not limited to phenoxy, naphthyloxy,and benzyloxy. Representative substituted aryloxy and arylalkoxy groupsmay be substituted one or more times with substituents such as thoselisted above.

The term “carboxylic acid” as used herein refers to a compound with a—C(O)OH group. The term “carboxylate” as used herein refers to a —C(O)O⁻group. A “protected carboxylate” refers to a —C(O)O-G where G is acarboxylate protecting group. Carboxylate protecting groups are wellknown to one of ordinary skill in the art. An extensive list ofprotecting groups for the carboxylate group functionality may be foundin Protective Groups in Organic Synthesis, Greene, T. W.; Wuts, P. G.M., John Wiley & Sons, New York, N.Y., (3rd Edition, 1999) which can beadded or removed using the procedures set forth therein and which ishereby incorporated by reference in its entirety and for any and allpurposes as if fully set forth herein.

The term “ester” as used herein refers to —COOR⁷⁰ groups. R⁷⁰ is asubstituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl,aralkyl, heterocyclylalkyl or heterocyclyl group as defined herein.

The term “amide” (or “amido”) includes C- and N-amide groups, i.e.,—C(O)NR⁷¹R⁷², and —NR⁷¹C(O)R⁷² groups, respectively. R⁷¹ and R⁷² areindependently hydrogen, or a substituted or unsubstituted alkyl,alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl orheterocyclyl group as defined herein. Amido groups therefore include butare not limited to carbamoyl groups (—C(O)NH₂) and formamide groups(—NHC(O)H). In some embodiments, the amide is —NR⁷¹C(O)—(C₁₋₅ alkyl) andthe group is termed “carbonylamino,” and in others the amide is—NHC(O)-alkyl and the group is termed “alkanoylamino.”

The term “nitrile” or “cyano” as used herein refers to the —CN group.

Urethane groups include N- and O-urethane groups, i.e., —NR⁷³C(O)OR⁷⁴and —OC(O)NR⁷³R⁷⁴ groups, respectively. R⁷³ and R⁷⁴ are independently asubstituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl,aralkyl, heterocyclylalkyl, or heterocyclyl group as defined herein. R⁷³may also be H.

The term “amine” (or “amino”) as used herein refers to —NR⁷⁵R⁷⁶ groups,wherein R⁷⁵ and R⁷⁶ are independently hydrogen, or a substituted orunsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl,heterocyclylalkyl or heterocyclyl group as defined herein. In someembodiments, the amine is alkylamino, dialkylamino, arylamino, oralkylarylamino. In other embodiments, the amine is NH₂, methylamino,dimethylamino, ethylamino, diethylamino, propylamino, isopropylamino,phenylamino, or benzylamino.

The term “sulfonamido” includes S- and N-sulfonamide groups, i.e.,—SO₂NR⁷⁸R⁷⁹ and —NR⁷⁸SO₂R⁷⁹ groups, respectively. R⁷⁸ and R⁷⁹ areindependently hydrogen, or a substituted or unsubstituted alkyl,alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl, orheterocyclyl group as defined herein. Sulfonamido groups thereforeinclude but are not limited to sulfamoyl groups (—SO₂NH₂). In someembodiments herein, the sulfonamido is —NHSO₂-alkyl and is referred toas the “alkylsulfonylamino” group.

The term “thiol” refers to —SH groups, while sulfides include —SR⁸⁰groups, sulfoxides include —S(O)R⁸¹ groups, sulfones include —SO₂R⁸²groups, and sulfonyls include —SO₂OR⁸³. R⁸⁰, R⁸¹, R⁸², and R⁸³ are eachindependently a substituted or unsubstituted alkyl, cycloalkyl, alkenyl,alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group asdefined herein. In some embodiments the sulfide is an alkylthio group,—S-alkyl.

The term “urea” refers to —NR⁸⁴—C(O)—NR⁸⁵R⁸⁶ groups. R⁸⁴, R⁸³, and R⁸⁶groups are independently hydrogen, or a substituted or unsubstitutedalkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclyl, orheterocyclylalkyl group as defined herein.

The term “amidine” refers to —C(NR⁸⁷)NR⁸⁸R⁸⁹ and —NR⁸⁷C(NR⁸⁸)R⁸⁹,wherein R⁸⁷, Ra⁸⁸, and R⁸⁹ are each independently hydrogen, or asubstituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, arylaralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.

The term “guanidine” refers to —NR⁹⁰C(NR⁹¹)NR⁹²R⁹³, wherein R⁹⁰, R⁹¹,R⁹² and R⁹³ are each independently hydrogen, or a substituted orunsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl,heterocyclyl or heterocyclylalkyl group as defined herein.

The term “enamine” refers to —C(R⁹⁴)═C(R⁹⁵)NR⁹⁶R⁹⁷ and—NR⁹⁴C(R⁹⁵)═C(R⁹⁶)R⁹⁷, wherein R⁹⁴, R⁹⁵, R⁹⁶ and R⁹⁷ are eachindependently hydrogen, a substituted or unsubstituted alkyl,cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl orheterocyclylalkyl group as defined herein.

The term “halogen” or “halo” as used herein refers to bromine, chlorine,fluorine, or iodine. In some embodiments, the halogen is fluorine. Inother embodiments, the halogen is chlorine or bromine.

The term “hydroxyl” as used herein can refer to —OH or its ionized form,—O⁻.

The term “imide” refers to —C(O)NR⁹⁸C(O)R⁹⁹, wherein R⁹⁸ and R⁹⁹ areeach independently hydrogen, or a substituted or unsubstituted alkyl,cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl orheterocyclylalkyl group as defined herein.

The term “imine” refers to —CR¹⁰⁰(NR¹⁰¹) and —N(CR¹⁰⁰R¹⁰¹) groups,wherein R¹⁰⁰ and R¹⁰¹ are each independently hydrogen or a substitutedor unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl,heterocyclyl or heterocyclylalkyl group as defined herein, with theproviso that R¹⁰⁰ and R¹⁰¹ are not both simultaneously hydrogen.

The term “nitro” as used herein refers to an —NO₂ group.

The term “trifluoromethyl” as used herein refers to —CF₃.

The term “trifluoromethoxy” as used herein refers to —OCF₃.

The term “azido” refers to —N₃.

The term “trialkyl ammonium” refers to a —N(alkyl)₃ group. Atrialkylammonium group is positively charged and thus typically has anassociated anion, such as halogen anion.

The term “trifluoromethyldiazirido” refers to

The term “isocyano” refers to —NC.

The term “isothiocyano” refers to —NCS.

The term “pentafluorosulfanyl” refers to —SF₅.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the likeinclude the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 atoms refers to groupshaving 1, 2, or 3 atoms. Similarly, a group having 1-5 atoms refers togroups having 1, 2, 3, 4, or 5 atoms, and so forth.

Pharmaceutically acceptable salts of compounds described herein arewithin the scope of the present technology and include acid or baseaddition salts which retain the desired pharmacological activity and isnot biologically undesirable (e.g., the salt is not unduly toxic,allergenic, or irritating, and is bioavailable). When the compound ofthe present technology has a basic group, such as, for example, an aminogroup, pharmaceutically acceptable salts can be formed with inorganicacids (such as hydrochloric acid, hydroboric acid, nitric acid, sulfuricacid, and phosphoric acid), organic acids (e.g., alginate, formic acid,acetic acid, benzoic acid, gluconic acid, fumaric acid, oxalic acid,tartaric acid, lactic acid, maleic acid, citric acid, succinic acid,malic acid, methanesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, and p-toluenesulfonic acid) or acidic amino acids (suchas aspartic acid and glutamic acid). When the compound of the presenttechnology has an acidic group, such as for example, a carboxylic acidgroup, it can form salts with metals, such as alkali and earth alkalimetals (e.g., Na⁺, Li⁺, K⁺, Ca²⁺, Mg²⁺, Zn²⁺), ammonia or organic amines(e.g. dicyclohexylamine, trimethylamine, triethylamine, pyridine,picoline, ethanolamine, diethanolamine, triethanolamine) or basic aminoacids (e.g., arginine, lysine and ornithine). Such salts can be preparedin situ during isolation and purification of the compounds or byseparately reacting the purified compound in its free base or free acidform with a suitable acid or base, respectively, and isolating the saltthus formed.

Those of skill in the art will appreciate that compounds of the presenttechnology may exhibit the phenomena of tautomerism, conformationalisomerism, geometric isomerism and/or stereoisomerism. As the formuladrawings within the specification and claims can represent only one ofthe possible tautomeric, conformational isomeric, stereochemical orgeometric isomeric forms, it should be understood that the presenttechnology encompasses any tautomeric, conformational isomeric,stereochemical and/or geometric isomeric forms of the compounds havingone or more of the utilities described herein, as well as mixtures ofthese various different forms.

“Tautomers” refers to isomeric forms of a compound that are inequilibrium with each other. The presence and concentrations of theisomeric forms will depend on the environment the compound is found inand may be different depending upon, for example, whether the compoundis a solid or is in an organic or aqueous solution. For example, inaqueous solution, quinazolinones may exhibit the following isomericforms, which are referred to as tautomers of each other:

As another example, guanidines may exhibit the following isomeric formsin protic organic solution, also referred to as tautomers of each other:

Because of the limits of representing compounds by structural formulas,it is to be understood that all chemical formulas of the compoundsdescribed herein represent all tautomeric forms of compounds and arewithin the scope of the present technology.

Stereoisomers of compounds (also known as optical isomers) include allchiral, diastereomeric, and racemic forms of a structure, unless thespecific stereochemistry is expressly indicated. Thus, compounds used inthe present technology include enriched or resolved optical isomers atany or all asymmetric atoms as are apparent from the depictions. Bothracemic and diastereomeric mixtures, as well as the individual opticalisomers can be isolated or synthesized so as to be substantially free oftheir enantiomeric or diastereomeric partners, and these stereoisomersare all within the scope of the present technology.

The compounds of the present technology may exist as solvates,especially hydrates. Hydrates may form during manufacture of thecompounds or compositions comprising the compounds, or hydrates may formover time due to the hygroscopic nature of the compounds. Compounds ofthe present technology may exist as organic solvates as well, includingDMF, ether, and alcohol solvates among others. The identification andpreparation of any particular solvate is within the skill of theordinary artisan of synthetic organic or medicinal chemistry.

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced by an identifying citation. Alsowithin this disclosure are Arabic numerals referring to referencedcitations, the full bibliographic details of which are providedimmediately preceding the claims. The disclosures of these publications,patents and published patent specifications are hereby incorporated byreference into the present disclosure to more fully describe the presenttechnology.

The Present Technology

Although targeted radiotherapy has been practiced for some time usingmacrocyclic complexes of radionuclides, the macrocycles currently in use(e.g., DOTA) generally form complexes of insufficient stability withradionuclides, particularly for radionuclides of larger size, such asactinium, radium, bismuth, and lead isotopes. Such instability resultsin dissociation of the radionuclide from the macrocycle, and thisresults in a lack of selectivity to targeted tissue, which also resultsin toxicity to non-targeted tissue.

The present technology provides new macrocyclic complexes that aresubstantially more stable than those of the conventional art. Thus,these new complexes can advantageously target cancer cells moreeffectively, with substantially less toxicity to non-targeted tissuethan complexes of the art. Moreover, the new complexes canadvantageously be produced at room temperature, in contrast to DOTA-typecomplexes, which generally require elevated temperatures (e.g., at least80° C.) for complexation with the radionuclide. The present technologyalso specifically employs alpha-emitting radionuclides instead of betaradionuclides. Alpha-emitting radionuclides are of much higher energy,and thus substantially more potent, than beta-emitting radionuclides.

Thus, in one aspect, a compound of Formula I is provided:

or a pharmaceutically acceptable salt thereof, wherein

-   -   Z¹ is H or —X¹—W²;    -   Z² is OH or NH—W³;    -   Z³ is H or W;    -   α is 0 or 1;    -   X¹ is O, NH, or S;    -   W² and W³ are each independently H, alkyl, cycloalkyl, alkenyl,        cycloalkenyl, alkynyl, aryl, heterocyclyl, heteroaryl,        —CH₂CH₂—(OCH₂CH₂)_(w)—R′ where w is 1, 2, 3, 4, 5, 6, 7, 8, 9,        or 10, or —CH₂CH₂—(OCH₂CH₂)_(x)—OR′ where x is 1, 2, 3, 4, 5, 6,        7, 8, 9, or 10, each of which may optionally be substituted with        one or more of halo, —N₃, —OR′, —CH₂CH₂—(OCH₂CH₂)_(y)—R′ where y        is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —CH₂CH₂—(OCH₂CH₂)_(z)—OR′        where z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —SR′, —OC(O)R′,        —C(O)OR′, —C(S)OR′, —S(O)R′, —SO₂R′, —SO₂(OR′), —SO₂NR′₂,        —P(O)(OR′)₂, —P(O)R′(OR′), —P(O)R′₂, —CN, —OCN, —SCN, —NCO,        —NCS, —NR′—NH₂, —N═C═N—R′, —SO₂Cl, —C(O)Cl, or an epoxide group;    -   W⁵ and W⁷ are each independently OH, NH₂, SH, alkyl, cycloalkyl,        alkenyl, cycloalkenyl, alkynyl, aryl, heterocyclyl, heteroaryl,        —CH₂CH₂—(OCH₂CH₂)_(w)—R′ where w is 1, 2, 3, 4, 5, 6, 7, 8, 9,        or 10, or —CH₂CH₂—(OCH₂CH₂)_(x)—OR′ where x is 1, 2, 3, 4, 5, 6,        7, 8, 9, or 10, each of which may optionally be substituted with        one or more of halo, —N₃, —OR′, —CH₂CH₂—(OCH₂CH₂)y_(x)-R′ where        y is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —CH₂CH₂—(OCH₂CH₂)_(z)—OR′        where z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —SR′, —OC(O)R′,        —C(O)OR′, —C(S)OR′, —S(O)R′, —SO₂R′, —SO₂(OR′), —SO₂NR′₂,        —P(O)(OR′)₂, —P(O)R′(OR′), —P(O)R′₂, —CN, —OCN, —SCN, —NCO,        —NCS, —NR′—NH₂, —N═C═N—R′, —SO₂Cl, —C(O)Cl, or an epoxide group;        and    -   R′ is independently at each occurrence H, halo, —N₃, C₁-C₆        alkyl, C₃-C₆ cycloalkyl, C₂-C₆ alkenyl, C₅-C₈ cycloalkenyl,        C₂-C₆ alkynyl, C₈-C₁₀ cycloalkynyl, C₅-C₆ aryl, heterocyclyl, or        heteroaryl.

Significantly, the uncomplexed form of Formula I can be complexed with aradionuclide, such as an alpha-emitting radionuclide, at roomtemperature (generally 18-30° C., or about or no more than 20° C., 25°C., or 30° C.) at high radiochemical yields, e.g., at least or greaterthan 90%, 95%, 97%, or 98%.

In a related aspect, a compound of Formula IA is provided

or a pharmaceutically acceptable salt thereof, wherein

-   -   M¹ is an alpha-emitting radionuclide;    -   Z¹ is H or —X¹—W²;    -   Z² is OH or NH—W³;    -   Z³ is H or W⁷;    -   α is 0 or 1;    -   X¹ is O, NH, or S;    -   W² and W³ are each independently H, alkyl, cycloalkyl, alkenyl,        cycloalkenyl, alkynyl, aryl, heterocyclyl, heteroaryl,        —CH₂CH₂—(OCH₂CH₂)_(w)—R′ where w is 1, 2, 3, 4, 5, 6, 7, 8, 9,        or 10, or —CH₂CH₂—(OCH₂CH₂)_(x)—OR′ where x is 1, 2, 3, 4, 5, 6,        7, 8, 9, or 10, each of which may optionally be substituted with        one or more of halo, —N₃, —OR′, —CH₂CH₂—(OCH₂CH₂)_(y)—R′ where y        is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —CH₂CH₂—(OCH₂CH₂)_(z)—OR′        where z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —SR′, —OC(O)R′,        —C(O)OR′, —C(S)OR′, —S(O)R′, —SO₂R′, —SO₂(OR′), —SO₂NR′₂,        —P(O)(OR′)₂, —P(O)R′(OR′), —P(O)R′₂, —CN, —OCN, —SCN, —NCO,        —NCS, —NR′—NH₂, —N═C═N—R′, —SO₂Cl, —C(O)Cl, or an epoxide group;    -   W⁵ and W⁷ are each independently OH, NH₂, SH, alkyl, cycloalkyl,        alkenyl, cycloalkenyl, alkynyl, aryl, heterocyclyl, heteroaryl,        —CH₂CH₂—(OCH₂CH₂)_(w)—R′ where w is 1, 2, 3, 4, 5, 6, 7, 8, 9,        or 10, or —CH₂CH₂—(OCH₂CH₂)_(x)—OR′ where x is 1, 2, 3, 4, 5, 6,        7, 8, 9, or 10, each of which may optionally be substituted with        one or more of halo, —N₃, —OR′, —CH₂CH₂—(OCH₂CH₂)y_(x)-R′ where        y is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —CH₂CH₂—(OCH₂CH₂)_(z)—OR′        where z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —SR′, —OC(O)R′,        —C(O)OR′, —C(S)OR′, —S(O)R′, —SO₂R′, —SO₂(OR′), —SO₂NR′₂,        —P(O)(OR′)₂, —P(O)R′(OR′), —P(O)R′₂, —CN, —OCN, —SCN, —NCO,        —NCS, —NR′—NH₂, —N═C═N—R′, —SO₂Cl, —C(O)Cl, or an epoxide group;        and    -   R′ is independently at each occurrence H, halo, —N₃, C₁-C₆        alkyl, C₃-C₆ cycloalkyl, C₂-C₆ alkenyl, C₅-C₈ cycloalkenyl,        C₂-C₆ alkynyl, C₈-C₁₀ cycloalkynyl, C₅-C₆ aryl, heterocyclyl, or        heteroaryl.

In any embodiment disclosed herein, it may be that M¹ is actinium-225(²²⁵Ac³⁺), radium-223 (²³³Ra²⁺), bismuth-213 (²¹³Bi³⁺), lead-212(²¹²Pb²⁺ and/or ²¹²Pb⁴⁺), terbium-149 (¹⁴⁹Tb³⁺), fermium-255 (²⁵⁵Fm³⁺),thorium-227 (²²⁷Th⁴⁺), thorium-226 (²²⁶Th⁴⁺), astatine-211 (²¹¹At⁺),astatine-217 (²¹⁷At⁺), or uranium-230.

In a further related aspect, the present technology provides a compounduseful in targeted radiotherapy of cancer and/or mammalian tissueoverexpressing prostate specific membrane antigen (“PSMA”) (a “targetingcompound”) where the compound is of Formula II

or a pharmaceutically acceptable salt thereof, wherein

-   -   M¹ is an alpha-emitting radionuclide;    -   Z¹ is H or -L³-R²²;    -   Z² is OH or NH-L⁴-R²⁴;    -   Z³ is H or -L⁶-R²⁸;    -   α is 0 or 1;    -   X¹ is O, NH, or S;    -   L³, L⁴, L⁵, and L⁶ are independently at each occurrence a bond        or a linker group; and    -   R²², R²⁴, R²⁶, and R²⁸ each independently comprises an antibody,        antibody fragment (e.g., an antigen-binding fragment), a binding        moiety, a binding peptide, a binding polypeptide (such as a        selective targeting oligopeptide containing up to 50 amino        acids), a binding protein, an enzyme, a nucleobase-containing        moiety (such as an oligonucleotide, DNA or RNA vector, or        aptamer), or a lectin.

In any embodiment disclosed herein encompassed by Formula II, M¹ may beactinium-225 (²²⁵Ac³⁺), radium-223 (²³³Ra²⁺), bismuth-213 (²¹³Bi³⁺),lead-212 (²¹²Pb²⁺ and/or ²¹²Pb⁴⁺), terbium-149 (¹⁴⁹Tb³⁺), fermium-255(²⁵⁵Fm³⁺), thorium-227 (²²⁷Th⁴⁺), thorium-226 (²²⁶Th⁴⁺), astatine-211(²¹¹At⁺), astatine-217 (²¹⁷At⁺), or uranium-230.

Representative R²², R²⁴, R²⁶, and R²⁸ groups include those antibodieslisted in Table A as well as antigen-binding fragments of suchantibodies and any equivalent embodiments, as would be known to those ofordinary skill in the art.

TABLE A Representative Antibodies Antibody Disclosed In (Trade Name(s))(U.S. Patent or Patent Appl. Publ. No.)* Belimumab 7,138,501 (Benlysta)Mogamulizumab 6,989,145 (Poteligeo) Blinatumomab 7,112,324 (Blincyto)Ibritumomab tiuxetan 5,776,456 (Zevalin) Obinutuzumab 6,602,684 (Gazyva)Ofatumumab¹ 8,529,902 (Arzerra) Rituximab 5,736,137 (Rituxan, MabThera)Inotuzumab ozogamicin 8,153,768 (Besponsa) Moxetumomab pasudotox8,809,502 (Lumoxiti) Brentuximab vedotin 7,829,531; 7,090,843 (Adcetris)Daratumumab 7,829,673 (Darzalex) Ipilimumab 6,984,720 (Yervoy) Cetuximab6,217,866 (Erbitux) Necitumumab 7,598,350 (Portrazza) Panitumumab6,235,883 (Vectibix) Dinutuximab² 7,432,357 (Unituxin) Pertuzumab7,862,817 (Perjeta, Omnitarg) Trastuzumab³ 5,821,337 (Herceptin)Trastuzumab emtansine 7,097,840 (Kadcyla) Siltuximab 7,612,182 (Sylvant)Cemiplimab⁴ 9,987,500 (Libtayo) Nivolumab 8,008,449 (Opdivo)Pembrolizumab 8,354,509 (Keytruda) Olaratumab 8,128,929 (Lartruvo)Atezolizumab 8,217,149 (Tecentriq) Avelumab⁵ 9,624,298 (Bavencio)Durvalumab 8,779,108 (Imfinzi) Capromab pendetide 5,162,504(Prostascint) Elotuzumab 7,709,610 (Empliciti) Denosumab 6,740,522(Prolia, Xgeva) Ziv-aflibercept 7,070,959 (Zaltrap) Bevacizumab6,054,297 (Avastin) Ramucirumab 7,498,414 (Cyramza) Tositumomab6,565,827; 6,287,537;, 6,090,365; (Bexxar) 6,015,542; 5,843,398;5,595,721 Gemtuzumab ozogamicin 5,773,001 (Mylotarg) Alemtuzumab6,569,430; 5,846,534 (Campath-1H) Cixutumumab 7,968,093; 7,638,605Girentuximab 8,466,263 (Rencarex) Nimotuzumab 6,506,883 (Theracim,Theraloc) Catumaxomab 9,017,676; 8,663,638; 2013/0309234A1 (Removab)Etaracizumab 2004/0001835A1 (Abegrin, Vitaxin) ¹Also designated 2F2.²Also designated Ch14.18. ³Also designated HuMaB4D5-8. ⁴Also designatedH4H7798N. ⁵Also designated A09-246-2. *Note: the disclosures of the eachof the patents and patent publications listed in Table A areincorporated herein by reference.

In any embodiment disclosed herein, it may be that the binding peptidecomprises comprises a prostate specific membrane antigen (“PSMA”)binding peptide, a somatostatin receptor agonist, a bombesin receptoragonist, a seprase binding compound, or a binding fragment thereof.Exemplary PSMA binding peptides include, but are not limited to, thoseaccording to the following structure

where nn is 0, 1, or 2, and P¹, P², and P³ are each independently H,methyl, benzyl, 4-methoxybenzyl, or tert-butyl. In any embodimentherein, it may be that each of P¹, P², and P³ are H.

Somatostatin, illustrated in Scheme A, is a peptide hormone thatregulates the endocrine system and affects neurotransmission and cellproliferation via interaction with G protein-coupled somatostatinreceptors and inhibition of the release of numerous secondary hormones.Somatostatin has two active forms produced by alternative cleavage of asingle preproprotein. There are five known somatostatin receptors, allbeing G protein-coupled seven transmembrane receptors: SST1 (SSTR1);SST2 (SSTR2); SST3 (SSTR3); SST4 (SSTR4); and SST5 (SSTR5). Exemplarysomatostatin receptor agonists include somatostatin itself, lanreotide,octreotate, octreotide, pasireotide, and vapreotide.

Many neuroendocrine tumors express SSTR2 and the other somatostatinreceptors. Long acting somatostatin agonists (e.g., Octreotide,Lanreotide) are used to stimulate the SSTR2 receptors, and thus toinhibit further tumor proliferation. See, Zatelli M C, et al., (April2007). “Control of pituitary adenoma cell proliferation by somatostatinanalogs, dopamine agonists and novel chimeric compounds”. EuropeanJournal of Endocrinology/European Federation of Endocrine Societies. 156Suppl 1: S29-35. Octreotide is an octapeptide that mimics naturalsomatostatin but has a significantly longer half-life in vivo.Octreotide is used for the treatment of growth hormone producing tumors(acromegaly and gigantism), when surgery is contraindicated, pituitarytumors that secrete thyroid stimulating hormone (thyrotropinoma),diarrhea and flushing episodes associated with carcinoid syndrome, anddiarrhea in people with vasoactive intestinal peptide-secreting tumors(VIPomas). Lanreotide is used in the management of acromegaly andsymptoms caused by neuroendocrine tumors, most notably carcinoidsyndrome. Pasireotide is a somatostatin analog with an increasedaffinity to SSTR5 compared to other somatostatin agonists and isapproved for treatment of Cushing's disease and acromegaly. Vapreotideis used in the treatment of esophageal variceal bleeding in patientswith cirrhotic liver disease and AIDS-related diarrhea.

Bombesin is a peptide originally isolated from the skin of the Europeanfire-bellied toad (Bombina bombina). In addition to stimulating gastrinrelease from G cells, bombesin activates at least three differentG-protein-coupled receptors: BBR1, BBR2, and BBR3, where such activityincludes agonism of such receptors in the brain. Bombesin is also atumor marker for small cell carcinoma of lung, gastric cancer,pancreatic cancer, and neuroblastoma. Bombesin receptor agonistsinclude, but are not limited to, BBR-1 agonists, BBR-2 agonists, andBBR-3 agonists. Seprase (or Fibroblast Activation Protein (FAP)) is anintegral membrane serine peptidase. In addition to gelatinase activity,seprase has a dual function in tumour progression. Seprase promotes cellinvasiveness towards the ECM and also supports tumour growth andproliferation. Seprase binding compounds include seprase inhibitors

In a further related aspect, a modified antibody, modified antibodyfragment, or modified binding peptide comprising a linkage arising fromconjugation of a compound of Formula I or pharmaceutically acceptablesalt thereof, with an antibody, antibody fragment, or binding peptide.In a related aspect, a modified antibody, modified antibody fragment, ormodified binding peptide is provided that includes a linkage arisingfrom conjugation of a compound of Formula IA or a pharmaceuticallyacceptable salt thereof, with an antibody, antibody fragment, or bindingpeptide. In any embodiment disclosed herein, it may be that the antibodyincludes belimumab, Mogamulizumab, Blinatumomab, Ibritumomab tiuxetan,Obinutuzumab, Ofatumumab, Rituximab, Inotuzumab ozogamicin, Moxetumomabpasudotox, Brentuximab vedotin, Daratumumab, Ipilimumab, Cetuximab,Necitumumab, Panitumumab, Dinutuximab, Pertuzumab, Trastuzumab,Trastuzumab emtansine, Siltuximab, Cemiplimab, Nivolumab, Pembrolizumab,Olaratumab, Atezolizumab, Avelumab, Durvalumab, Capromab pendetide,Elotuzumab, Denosumab, Ziv-aflibercept, Bevacizumab, Ramucirumab,Tositumomab, Gemtuzumab ozogamicin, Alemtuzumab, Cixutumumab,Girentuximab, Nimotuzumab, Catumaxomab, or Etaracizumab. In anyembodiment disclosed herein, it may be that the antibody fragmentincludes an antigen-binding fragment of belimumab, Mogamulizumab,Blinatumomab, Ibritumomab tiuxetan, Obinutuzumab, Ofatumumab, Rituximab,Inotuzumab ozogamicin, Moxetumomab pasudotox, Brentuximab vedotin,Daratumumab, Ipilimumab, Cetuximab, Necitumumab, Panitumumab,Dinutuximab, Pertuzumab, Trastuzumab, Trastuzumab emtansine, Siltuximab,Cemiplimab, Nivolumab, Pembrolizumab, Olaratumab, Atezolizumab,Avelumab, Durvalumab, Capromab pendetide, Elotuzumab, Denosumab,Ziv-aflibercept, Bevacizumab, Ramucirumab, Tositumomab, Gemtuzumabozogamicin, Alemtuzumab, Cixutumumab, Girentuximab, Nimotuzumab,Catumaxomab, or Etaracizumab. In any embodiment disclosed herein, it maybe that the binding peptide includes a prostate specific membraneantigen (“PSMA”) binding peptide, a somatostatin receptor agonist, abombesin receptor agonist, a seprase binding compound, or a bindingfragment thereof.

As an example of a modified antibody, modified antibody fragment, ormodified binding peptide of the present technology, it may be that thelinkage is a thiocynate linkage; wherein the thiocyanate linkage arisesfrom conjugation of the compound with the antibody, antibody fragment,or binding peptide; and wherein the compound is

or pharmaceutically acceptable salt thereof.

As another example of a modified antibody, modified antibody fragment,or modified binding peptide of the present technology, it may be thatthe linkage is a thiocynate linkage; wherein the thiocyanate linkagearises from conjugation of the compound with the antibody, antibodyfragment, or binding peptide; and wherein the compound is

or a pharmaceutically acceptable salt thereof.

In any embodiment herein, it may be that the structures includecompounds of Formula III, a modified antibody, modified antibodyfragment, or modified binding peptide comprising a linkage arising fromconjugation of a compound of Formula III or pharmaceutically acceptablesalt thereof, with an antibody, antibody fragment, or binding peptide,compounds of Formula IV, a modified antibody, modified antibodyfragment, or modified binding peptide comprising a linkage arising fromconjugation of a compound of Formula IV or pharmaceutically acceptablesalt thereof, with an antibody, antibody fragment, or binding peptide,and targeting compounds of Formula V

or a pharmaceutically acceptable salt thereof,

or a pharmaceutically acceptable salt thereof,

or a pharmaceutically acceptable salt thereof, wherein M² isindependently at each occurrence an alpha-emitting radionuclide.

Targeting compounds of Formula V may be prepared by a process thatincludes reacting a compound of Formula III or IV with R²²—W¹, whereTable B provides representative examples (where n is independently ateach occurrence 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). As such, R²² may beconjugated to macrocycle R²¹ by reaction of complementary chemicalfunctional groups W¹ and W² to form linker L³. For example, R²²—W¹ mayinclude a modified target amino acid residue within a protein (e.g., oneof the representative antibodies disclosed in Table A or anantigen-binding fragment thereof; a PSMA binding peptide, a somatostatinreceptor agonist, a bombesin receptor agonist, a seprase bindingcompound, or a binding fragment of any one thereof). W¹ may include areactive chemical functional moiety, non-limiting examples of which aredisclosed in the Table B, where W² may be selected to selectively reactwith W¹ in order to provide L³ of Formula V.

TABLE B

Final Conjugation Product W¹—R²² R²¹—X¹—W² X¹ (R²¹—X¹—L³—R²²) N₃—R²²

NH

and/or

O

and/or

NH

and/or

O

and/or

NH

and/or

O

and/or

NH

O

S

NH

O

S

and/or

NH

and/or

O

and/or

NH

O

H₂N—R²²

NH

O

NH

O

S

NH

and/or

O

and/or

NH

and/or

O

and/or

S

and/or

NH

O

NH

O

NH

O

NH

O

In any embodiment herein, it may be that the structures includecompounds of Formula VI, a modified antibody, modified antibodyfragment, or modified binding peptide comprising a linkage arising fromconjugation of a compound of Formula VI or pharmaceutically acceptablesalt thereof, with an antibody, antibody fragment, or binding peptide,compounds of Formula VII, a modified antibody, modified antibodyfragment, or modified binding peptide comprising a linkage arising fromconjugation of a compound of Formula VII or pharmaceutically acceptablesalt thereof, with an antibody, antibody fragment, or binding peptide,and targeting compounds of Formula VIII

or a pharmaceutically acceptable salt thereof,

or a pharmaceutically acceptable salt thereof,

or a pharmaceutically acceptable salt thereof, wherein M³ isindependently at each occurrence an alpha-emitting radionuclide.

Targeting compounds of Formula VIII may be prepared by a process thatincludes reacting a compound of Formula VI or VII with R²⁴—W⁴, whereTable C provides representative examples (where n is independently ateach occurrence 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). As such, R²⁴ may beconjugated to macrocycle R²³ by reaction of complementary chemicalfunctional groups W³ and W⁴ to form linker L⁴. For example, R²⁴—W⁴ mayinclude a modified target amino acid residue within a protein (e.g., oneof the representative antibodies disclosed in Table A or anantigen-binding fragment thereof; a PSMA binding peptide, a somatostatinreceptor agonist, a bombesin receptor agonist, a seprase bindingcompound, or a binding fragment of any one thereof). W⁴ may include areactive chemical functional moiety, non-limiting examples of which aredisclosed in the Table C, where W³ may be selected to selectively reactwith W⁴ in order to provide L⁴ of Formula VIII.

TABLE C

Final Conjugation Product R²³—W³ W⁴—R²⁴ R²³—L⁴—R²⁴

and/or

  and/or  

H₂N—R²⁴

N₃—R²⁴

  and/or

H₂N—R²⁴

In any embodiment herein, it may be that the structures includecompounds of Formula IX, a modified antibody, modified antibodyfragment, or modified binding peptide comprising a linkage arising fromconjugation of a compound of Formula IX or pharmaceutically acceptablesalt thereof, with an antibody, antibody fragment, or binding peptide,compounds of Formula X, a modified antibody, modified antibody fragment,or modified binding peptide comprising a linkage arising fromconjugation of a compound of Formula X or pharmaceutically acceptablesalt thereof, with an antibody, antibody fragment, or binding peptide,and targeting compounds of Formula XI

or a pharmaceutically acceptable salt thereof,

or a pharmaceutically acceptable salt thereof,

or a pharmaceutically acceptable salt thereof, wherein M⁴ isindependently at each occurrence an alpha-emitting radionuclide.

Targeting compounds of Formula XI may be prepared by a process thatincludes reacting a compound of Formula IX or X with R²⁶—W⁶, where TableD provides representative examples (where n is independently at eachoccurrence 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). As such, R²⁶ may beconjugated to macrocycle R²⁵ by reaction of complementary chemicalfunctional groups W⁵ and W⁶ to form linker L⁵. For example, R²⁶—W⁶ mayinclude a modified target amino acid residue within a protein (e.g., oneof the representative antibodies disclosed in Table A or anantigen-binding fragment thereof; a PSMA binding peptide, a somatostatinreceptor agonist, a bombesin receptor agonist, a seprase bindingcompound, or a binding fragment of any one thereof). W⁶ may include areactive chemical functional moiety, non-limiting examples of which aredisclosed in the Table D, where W⁵ may be selected to selectively reactwith W⁶ in order to provide L⁵ of Formula IX.

TABLE D

R²⁵—W⁵ W⁶—R²⁶ Final Conjugation Product

H₂N—R²⁶

and/or

and/or

In any embodiment herein, it may be that the structures includecompounds of Formula XII, a modified antibody, modified antibodyfragment, or modified binding peptide comprising a linkage arising fromconjugation of a compound of Formula XII or pharmaceutically acceptablesalt thereof, with an antibody, antibody fragment, or binding peptide,compounds of Formula XIII, a modified antibody, modified antibodyfragment, or modified binding peptide comprising a linkage arising fromconjugation of a compound of Formula XIII or pharmaceutically acceptablesalt thereof, with an antibody, antibody fragment, or binding peptide,and targeting compounds of Formula XIV

or a pharmaceutically acceptable salt thereof,

or a pharmaceutically acceptable salt thereof,

or a pharmaceutically acceptable salt thereof, wherein M⁵ isindependently at each occurrence an alpha-emitting radionuclide.

Targeting compounds of Formula XIV may be prepared by a process thatincludes reacting a compound of Formula XII or XIII with R²⁸—W⁸, whereTable E provides representative examples (where n is independently ateach occurrence 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). As such, R²⁸ may beconjugated to macrocycle R²⁷ by reaction of complementary chemicalfunctional groups W⁷ and W⁸ to form linker L⁴. For example, R²⁸—W⁸ mayinclude a modified target amino acid residue within a protein (e.g., oneof the representative antibodies disclosed in Table A or anantigen-binding fragment thereof; a PSMA binding peptide, a somatostatinreceptor agonist, a bombesin receptor agonist, a seprase bindingcompound, or a binding fragment of any one thereof). W⁸ may include areactive chemical functional moiety, non-limiting examples of which aredisclosed in the Table E, where W⁷ may be selected to selectively reactwith W⁸ in order to provide L⁶ of Formula XIV.

TABLE E

R²⁷—W⁷ W⁸—R²⁸ Final Conjugation Product

H₂N—R²⁸

and/or

A person of ordinary skill in the art will recognize that numerouschemical conjugation strategies provide ready access to targetingcompounds of the present technology, whereby exposed amino acid residueson a protein (e.g., an antibody) undergo well-known reactions withreactive moieties on a prosthetic molecule. For example, amide couplingis a well-known route, where—as an example—lysine residues on theantibody surface react with terminal activated carboxylic acid esters togenerate stable amide bonds. Amide coupling is typically mediated by anyof several coupling reagents (e.g., HATU, EDC, DCC, HOBT, PyBOP, etc.),which are detailed elsewhere. (See generally Eric Valeur & Mark Bradley,Amide Bond Formation: Beyond the Myth of Coupling Reagents, 38 CHEM.SOC. REV. 606 (2009).) These and other amide coupling strategies aredescribed in a recent review by Tsuchikama. (Kyoji Tsuchikama & ZhiqiangAn, Antibody-Drug Conjugates: Recent Advances in Conjugation and LinkerChemistries, 9 PROTEIN CELL 33, 36 (2018); see also, e.g., A. C. Lazaret al., Analysis of the Composition of Immunoconjugates UsingSize-Exclusion Chromatography Coupled to Mass Spectrometry, 19 RAPIDCOMMUN. MASS SPECTROM. 1806 (2005).)

Additionally, a person of ordinary skill in the art will recognize thatcysteine coupling reactions may be employed to conjugate prostheticmolecules with thiol-reactive termini to protein surfaces throughexposed thiol side chains on cysteine residues on the protein (e.g.,antibody) surface. (See generally Tsuchikama & An, supra, at 36-37; seealso, e.g., Pierre Adumeau et al., Thiol-Reactive Bifunctional Chelatorsfor the Creation of Site-Selectively Modified Radioimmunoconjugates withImproved Stability, 29 BIOCONJUGATE CHEM. 1364 (2018).) Because cysteineresidues readily form disulfide linkages with nearby cysteine residuesunder physiological conditions, rather than existing as free thiols,some cysteine coupling strategies may rely upon selective reduction ofdisulfides to generate a higher number of reactive free thiols. (Seeid.) Cysteine coupling techniques known in the art include, but are notlimited to, cys alkylation reactions, cysteine rebridging reactions, andcys-aryl coupling using organometallic palladium reagents. (See, e.g.,C. R. Behrens et al., Antibody-Drug Conjugates (ADCs) Derived fromInterchain Cysteine Cross-Linking Demonstrates Improved Homogeneity andOther Pharmacological Properties Over Conventional Heterogeneous ADCs,12 MOL. PHARM. 3986 (2015); Vinogradova et al., Organometallic PalladiumReagents for Cysteine Bioconjugation, 526 NATURE 687 (2015); see alsoTsuchikama, supra, at 37 (collecting examples).)

Protein conjugation strategies using non-natural amino acid side chainsare also well-known in the art. For example, “click chemistries” provideaccess to conjugated proteins, by rapid and selective chemicaltransformations under a diverse range of reaction conditions. Clickchemistries are known to yield peptide conjugates with limitedby-product formation, despite the presence of unprotected functionalgroups, in aqueous conditions. One important non-limiting example of aclick reaction in the formation of conjugated peptides is thecopper(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition reaction(CuAAC). (See Liyuan Liang & Didier Astruc, The Copper(I)-CatalysedAlkyne-Azide Cycloaddition (CuAAC) “Click” Reaction and ItsApplications: An Overview, 255 COORD. CHEM. REV. 2933 (2011); see also,e.g., Herman S. Gill & Jan Marik, Preparation of ¹⁸ F-labeled Peptidesusing the Copper(I)-Catalyzed Azide-Alkyne 1,3-Dipolar Cycloaddition, 6NATURE PROTOCOLS 1718 (2011).) The CuAAC click reaction may be carriedout in the presence of ligands to enhance reaction rates. Such ligandsmay include, for example, polydentate nitrogen donors, including amines(e.g., tris(triazolyl)methyl amines) and pyridines. (See Liang & Astruc,supra, at 2934 (collecting examples); P. L. Golas et al., 39MACROMOLECULES 6451 (2006).) Other widely-utilized click reactionsinclude, but are not limited to, thiol-ene, oxime, Diels-Alder, Michaeladdition, and pyridyl sulfide reactions.

Copper-free (Cu-free) click methods are also known in the art fordelivery of therapeutic and/or diagnostic agents, such as radionuclides(e.g., ¹⁸F), chemotherapeutic agents, dyes, contrast agents, fluorescentlabels, chemiluminescent labels, or other labels, to protein surfaces.Cu-free click methods may permit stable covalent linkage between targetmolecules and prosthetic groups. Cu-free click chemistry may includereacting an antibody or antigen-binding fragment, which has beenmodified with a non-natural amino acid side chain that includes anactivating moiety such as a cyclooctyne (e.g., dibenzocyclooctyne(DBCO)), a nitrone or an azide group, with a prosthetic group thatpresents a corresponding or complementary reactive moiety, such as anazide, nitrone or cyclooctyne (e.g., DBCO). (See, e.g., David. J.Donnelly et al., Synthesis aid Biologic Evaluation of a Novel ¹⁸F-Labeled Adnectin as a PET Radioligand for Imaging PD-L1 Expression, 59J. NUCL. MED. 529 (2018).) For example, where the targeting moleculecomprises a cyclooctyne, the prosthetic group may include an azide,nitrone, or similar reactive moiety. Where the targeting moleculeincludes an azide or nitrone, the prosthetic group may present acomplementary cyclooctyne, alkyne, or similar reactive moiety. Cu-freeclick reactions may be carried out at room temperature, in aqueoussolution, in the presence of phosphate-buffered saline (PBS). Theprosthetic group may be radiolabeled (e.g., with ¹⁸F) or may beconjugated to any alternative diagnostic and/or therapeutic agent (e.g.,a chelating agent). (See id. at 531.)

The compounds of any embodiment and aspect herein of the presenttechnology may be a tripartite compound. However, such tripartitecompounds are not restricted to compositions including Formulas I, IA,or II. Thus, in an aspect, a tripartite compound is provided thatincludes a first domain that has relatively low but still specificaffinity for serum albumin (e.g., 0.5 to 50×10⁻⁶M), a second domainincluding a chelating moiety such as but not limited to those describedherein, and a third domain that includes tumor targeting moiety (TTT)having relatively high affinity for a tumor antigen (e.g., 0.5 to50×10⁻⁹M). The following exemplary peptide receptors, enzymes, celladhesion molecules, tumor associated antigens, growth factor receptorsand cluster of differentiation antigens are useful targets forconstructing the TTT domain: somatostatin peptide receptor-2 (SSTR2),gastrin-releasing peptide receptor, seprase (FAP-alpha), incretinreceptors, glucose-dependent insulinotropic polypeptide receptors,VIP-1, NPY, folate receptor, LHRH, and αvβ3, an overexpressed peptidereceptor, a neuronal transporter (e.g., noradrenaline transporter(NET)), or other tumor associated proteins such as EGFR, HER-2, VGFR,MUC-1, CEA, MUC-4, ED2, TF-antigen, endothelial specific markers,neuropeptide Y, uPAR, TAG-72, CCK analogs, VIP, bombesin, VEGFR,tumor-specific cell surface proteins, GLP-1, CXCR4, Hepsin, TMPRSS2,caspaces, Alpha V beta six, cMET. Other such targets will be apparent tothose of skill in the art, and compounds that bind these can beincorporated in the TTT to produce a tripartite radiotherapeuticcompound.

The following Formulas L-LIV provide exemplary general structures fortripartite compounds of the present technology.

where

-   -   TTT is independently at each occurrence a binding domain for a        somatostatin peptide receptor-2 (SSTR2), a gastrin-releasing        peptide receptor, a seprase (FAP-alpha), an incretin receptor, a        glucose-dependent insulinotropic polypeptide receptor, VIP-1,        NPY, a folate receptor, LHRH, αvβ3, an overexpressed peptide        receptor, a neuronal transporter (e.g., noradrenaline        transporter (NET)), a receptor for a tumor associated protein        (such as EGFR, HER-2, VGFR, MUC-1, CEA, MUC-4, ED2, TF-antigen,        endothelial specific markers, neuropeptide Y, uPAR, TAG-72, CCK        analogs, VIP, bombesin, VEGFR, tumor-specific cell surface        proteins, GLP-1, CXCR4, Hepsin, TMPRSS2, caspaces, Alpha V beta        six, cMET, or combination of any two or more thereof), or a        combination of any two or more thereof;    -   X⁵⁰¹ is independently at each occurrence absent, O, S, or NH;    -   L⁵⁰¹ is independently at each occurrence absent, —C(O)—,        —C(O)—NR⁴—, —C(O)—NR⁵-C₁-C₁₂ alkylene-, —C₁-C₁₂ alkylene-C(O)—,        —C(O)—NR⁶—C₁-C₁₂ alkylene-C(O)—, -arylene-,        —O(CH₂CH₂O)_(r)—CH₂CH₂C(O)—, —O(CH₂CH₂O)_(rr)—CH₂CH₂C(O)—NH—,        —O(CH₂CH₂O)_(rrr)—CH₂CH₂—, an amino acid, a peptide of 2, 3, 4,        5, 6, 7, 8, 9, or 10 amino acids, or a combination of any two or        more thereof, where r is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9, rr is        0, 1, 2, 3, 4, 5, 6, 7, 8, or 9, rrr is 0, 1, 2, 3, 4, 5, 6, 7,        8, or 9, and where R⁴, R⁵, and R⁶ are each independently H,        alkyl, or aryl;    -   Rad is independently at each occurrence a moiety capable of        including a radionuclide, optionally further including a        radionuclide;    -   L⁵⁰² is independently at each occurrence absent, —C(O)—,        —(CH₂CH₂O)_(s)—CH₂CH₂C(O)—, —(CH₂CH₂O)_(ss)—CH₂CH₂C(O)—NH—,        —(CH₂CH₂O)_(sss)—CH₂CH₂—, an amino acid, —CH(CO₂H)—(CH₂)₄—,        —CH(CO₂H)—(CH₂)₄—NH—, a peptide of 2, 3, 4, 5, 6, 7, 8, 9, 10,        11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids, or a        combination of any two or more thereof, where s is 0, 1, 2, 3,        4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19, ss        is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,        18, or 19, and sss is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,        13, 14, 15, 16, 17, 18, or 19;    -   Alb is independently at each occurrence an albumin-binding        moiety;    -   p is independently at each occurrence 0, 1, 2, or 3; and    -   q is independently at each occurrence 1 or 2.

In any embodiment disclosed herein, the radionuclide may be ¹⁷⁷Lu³⁺,¹⁷⁵Lu³⁺, ⁴⁵Sc³⁺, ⁶⁶Ga³⁺, ⁶⁷Ga³⁺, ⁶⁸Ga³⁺, ⁶⁹Ga³⁺, ⁷¹Ga³⁺, ⁸⁹Y³⁺, ⁸⁶Y³⁺,⁸⁹Zr⁴⁺, ⁹⁰Y³⁺, ^(99m)Tc⁺¹, ¹¹¹In³⁺, ¹¹³In⁺¹, ¹¹⁵In³⁺, ¹³⁹La³⁺, ¹³⁶Ce³⁺,¹¹⁸Ce³⁺, ¹⁴⁰Ce³⁺, ¹⁴²Ce³⁺, ¹⁵¹Eu³⁺, ¹⁵³Eu³⁺, ¹⁵²Dy³⁺, ¹⁴⁹Tb³⁺, ¹⁵⁹Tb³⁺,¹⁵⁴Gd³⁺, ¹⁵⁵Gd³⁺, ¹⁵⁶Gd³⁺, ¹⁵⁷Gd³⁺, ¹⁵⁸Gd³⁺, ¹⁶⁰Gd³⁺, ¹⁸⁸Re⁺¹, ¹⁸⁶Re⁺¹,213Bi³⁺, 211At⁺, ²¹⁷At⁺, ²²⁷Th⁴⁺, ²²⁶Th⁴⁺, ²²⁵Ac³⁺, ²³³Ra²⁺, ¹⁵²Dy³⁺,²¹³Bi³⁺, ²¹²Bi³⁺, ²¹¹Bi³⁺, ²¹²Pb²⁺, ²¹²Pb⁴⁺, ²⁵⁵Fm³⁺, or uranium-230.For example, the radionuclide may be an alpha-emitting radionuclide suchas ²¹³Bi³⁺, ²¹¹At⁺, ²²⁵Ac³⁺, ¹⁵²Dy³⁺, ²¹²Bi³⁺, ²¹¹Bi³⁺, ²¹⁷At⁺, ²²⁷Tb⁴⁺,²²⁶Th⁴⁺, ²³³Ra²⁺, ²¹²Pb²⁺, or ²¹²Pb⁴⁺.

In any embodiment disclosed herein, it may be the tripartite compoundsof Formulas L-LIV are of Formulas LV-LIX

where L⁵⁰³ is independently at each occurrence absent, —C(O)—, —C₁-C₁₂alkylene-, —C₁-C₁₂ alkylene-C(O)—, —C₁-C₂ alkylene-NR¹⁰—, -arylene-,—(CH₂CH₂O)_(z)—CH₂CH₂C(O)—, —(CH₂CH₂O)_(zz)—CH₂CH₂C(O)—NH—,—(CH₂CH₂O)_(zzz)—CH₂CH₂—, an amino acid, —CH(CO₂H)—(CH₂)₄—,—CH(CO₂H)—(CH₂)₄—NH—, a peptide of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 amino acids, or a combination of anytwo or more thereof, where z is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, or 19, zz is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, or 19, and zzz is 0, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19; and CHEL isindependently at each occurrence a covalently conjugated chelator thatoptionally includes a chelated radionuclide.

The albumin-binding moiety plays a role in modulating the rate of bloodplasma clearance of the compounds in a subject, thereby increasingcirculation time and compartmentalizing the cytotoxic action ofcytotoxin-containing domain and/or imaging capability of the imagingagent-containing domain in the plasma space instead of normal organs andtissues that may express antigen. Without being bound by theory, thiscomponent of the structure is believed to interact reversibly with serumproteins, such as albumin and/or cellular elements. The affinity of thisalbumin-binding moiety for plasma or cellular components of the bloodmay be configured to affect the residence time of the compounds in theblood pool of a subject. In any embodiment herein, the albuminbinding-moiety may be configured so that it binds reversibly ornon-reversibly with albumin when in blood plasma. In any embodimentherein, the albumin binding-moiety may be selected such that the bindingaffinity of the compound with human serum albumin is about 5 μM to about15 μM.

By way of example, the albumin-binding moiety of any embodiment hereinmay include a short-chain fatty acid, medium-chain chain fatty acid, along-chain fatty acid, myristic acid, a substituted or unsubstitutedindole-2-carboxylic acid, a substituted or unsubstituted4-oxo-4-(5,6,7,8-tetrahydronaphthalen-2-yl)butanoic acid, a substitutedor unsubstituted naphthalene acylsulfonamide, a substituted orunsubstituted diphenylcyclohexanol phosphate ester, a substituted orunsubstituted 2-(4-iodophenyl)acetic acid, a substituted orunsubstituted 3-(4-iodophenyl)propionic acid, or a substituted orunsubstituted 4-(4-iodophenyl)butanoic acid. Certain representativeexamples of albumin-binding moieties that may be included in anyembodiment herein include one or more of the following:

In any embodiment herein, the tripartite compounds may include analbumin-binding moiety that is

where Y⁵⁰¹, Y⁵⁰², Y⁵⁰³, Y⁵⁰⁴, and Y⁵⁰⁵ are independently H, halo, oralkyl, X⁵⁰³, X⁵⁰⁴, X⁵⁰⁵, and X⁵⁰⁶, are each independently O or S, aa isindependently at each occurrence 0, 1, or 2, bb is independently at eachoccurrence 0 or 1, cc is independently at each occurrence 0 or 1, and ddis independently at each occurrence 0, 1, 2, 3, or 4. In any embodimentherein, it may be that bb and cc cannot be the same value. In anyembodiment herein, it may be that Y⁵⁰³ is I and each of Y⁵⁰¹, Y⁵⁰²,Y⁵⁰³, Y⁵⁰⁴, and Y⁵⁰⁵ are each independently H.

Representative chelators useful in any embodiment of the presenttechnology include, but are not limited to, a covalently conjugatedsubstituted or unsubstituted chelator of the following group:

-   -   1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA),    -   1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA),    -   p-SCN-Bn-DOTA (also known as 2B-DOTA-NCS),    -   PIP-DOTA,    -   diethylenetriaminepentaacetic acid (DTPA),    -   PIP-DTPA,    -   AZEP-DTPA,    -   ethylenediamine tetraacetic acid (EDTA),    -   triethylenetetraamine-N,N,N′,N″,N″′,N″″-hexa-acetic acid (TTHA),    -   7-[2-(bis-carboxymethylamino)-ethyl]-4,10-bis-carboxymethyl-1,4,7,10-tetraaza-cyclododec-1-yl-acetic        acid (DEPA),    -   2,2′,2″-(10-(2-(bis(carboxymethyl)amino)-5-(4-isothiocyanatophenyl)        pentyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic        acid (3p-C-DEPA-NCS),    -   NETA,    -   {4-carboxymethyl-7-[2-(carboxymethylamino)-ethyl]-perhydro-1,4,7-triazonin-1-yl}-acetic        acid (NPTA),    -   diacetylpyridinebis(benzoylhydrazone),    -   1,4,7,10,13,16-hexaazacyclooctadecane-N,N′,N″,N″′,N″″,N″″′-hexaaceticacid        (HEHA), octadentate terephthalamide ligands,    -   siderophores,    -   2,2′-(4-(2-(bis(carboxymethyl)amino)-5-(4-isothiocyanatophenyl)pentyl)-10-(2-(bis(carboxymethyl)amino)ethyl)-1,4,7,10-tetraazacyclododecane-1,7-diyl)diacetic        acid,    -   N,N′-bis[(6-carboxy-2-pyridil)methyl]-4,13-diaza-18-crown-6        (H₂macropa),    -   6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)-4-isothiocyanatopicolinic        acid (macropa-NCS),    -   1,4,7,10-tetraaza-1,4,7,10-tetra(2-carbamonyl        methyl)cyclododecane (TCMC),    -   S-2-(4-Isothiocyanatobenzyl)-1,4,7,10-tetraaza-1,4,7,10-tetra(2-carbamoylmethyl)cyclododecane        (S-p-SCN-Bn-TCMC),    -   R-2-(4-Isothiocyanatobenzyl)-1,4,7,10-tetraaza-1,4,7,10-tetra(2-carbamoylmethyl)cyclododecane        (R-p-SCN-Bn-TCMC), and    -   3,9-carboxymethyl-6-(2-methoxy-5-isothiocyanatophenyl)carboxymethyl-3,6,9,15-tetraazabicyclo-[9.3.1]pentadeca-1(15),11,13-triene.

Certain members of this exemplary group are illustrated below.

It is to be understood that a “covalently conjugated” chelator means achelator (such as those listed above) wherein one or more bonds to ahydrogen atom contained therein are replaced by a bond to an atom of theremainder of the Rad and/or CHEL moiety, to L⁵⁰¹, and/or to L⁵⁰², or api bond between two atoms is replaced by a bond from one of the twoatoms to an atom of the remainder of the Rad and/or CHEL moiety, toL⁵⁰¹, and/or to L⁵⁰², and the other of the two atoms includes a newbond, e.g. to a hydrogen (such as reaction of an —NCS group in thechelator to provide the covalently conjugated chelator).

In any embodiment disclosed herein, it may be that the CHEL of thetripartite compounds is a chelator as provided in the compounds ofFormula I, IA, or II. For example, tripartite compound may be atargeting compound of Formula II where R²², R²⁴, R²⁶, and R²⁸ are eachindependently

In any embodiment disclosed herein, TTT may be

where

-   -   W⁵⁰¹ is —C(O)—, —(CH₂)_(ww)—, or —(CH₂)_(oo)—NH—C(O)—;    -   mm is 0 or 1;    -   ww is 1 or 2;    -   oo is 1 or 2; and    -   P⁵⁰¹, P⁵⁰², and P⁵⁰³ are each independently H, methyl, benzyl,        4-methoxybenzyl, or tert-butyl.

In any embodiment herein, it may be that each of P⁵⁰¹, P⁵⁰², and P⁵⁰³are H.

The tripartite compounds of the present technology include variations onany of the three domains: e.g., the domain including the chelator, thedomain including the albumin-binding group, or the domain including thetumor targeting moiety. The following are exemplary.

RPS-092.

In any embodiment disclosed herein, RPS-92 may optionally chelate²¹³Bi³⁺, ²¹¹At⁺, ²²⁵Ac³⁺, ¹⁵²Dy³⁺, ²¹²Bi³⁺, ²¹¹Bi³⁺, ²¹⁷At⁺, ²²⁷Th⁴⁺,²²⁶Th⁴⁺, ²³³Ra²⁺, ²¹²Pb²⁺, or ²¹²Pb⁴⁺.

NTI-093 is an analog of NTI-063, where TCMC is used as the chelator.

In any embodiment disclosed herein, NTI-93 may optionally chelate¹¹²Pb²⁺ or ²¹²Pb⁴⁺.

NTI-094 is an analog of NTI-072, where TCMC is used as the chelator.

In any embodiment disclosed herein, NTI-94 may optionally chelate²¹²Pb²⁺ or ²¹²Pb⁴⁺.

The following is a Bromo analog of NTI-063, with modification to thealbumin binding domain.

The following is a Chloro analog of NTI-063, with modification to thealbumin binding domain.

NTI-309 modifies the tumor targeting domain, to target seprase(Fibroblast Activation Protein/FAP).

The NTI-309 compound can be include TCMC as the chelator.

In any embodiment disclosed herein, NTI-309 may optionally chelate²¹²Pb²⁺ or ²¹²Pb⁴⁺.

The following is a Boronic acid analog of NTI-309.

The following is a Boronic acid analog of NTI-309, using TCMC as achelator.

In any embodiment disclosed herein, this analog may optionally chelate²¹²Pb²⁺ or ²¹²Pb⁴⁺.

Further by way of specific examples, a derivative of RPS-072 (whichitself targets PSMA), can be constructed, where TTT has affinity for theSSTR2 receptor, using a derivative of lanreotide where this compound (A)has a molecular weight of 3537.93 and a formula of C₁₆₅H₂₃₅IN₂₅O₄₄S₃.Similarly, a derivative of RPS-072 can be prepared, that targetsGRP/bombesin receptor, where this compound (B) has a molecular weight of3537.93 and a formula of C₁₆₇H₂₄₈IN₃₁O₄₄S.

The present technology also provides compositions (e.g., pharmaceuticalcompositions) and medicaments comprising any of one of the embodimentsof the compounds of Formulas I, IA, II, any one of the modifiedantibodies, modified antibody fragments, or modified binding peptides ofthe present technology disclosed herein, or any one of the embodimentsof the tripartite compounds disclosed herein and a pharmaceuticallyacceptable carrier or one or more excipients or fillers (collectivelyreferred to as “pharmaceutically acceptable carrier” unless otherwisespecified). The compositions may be used in the methods and treatmentsdescribed herein. The pharmaceutical composition may include aneffective amount of any embodiment of the compounds of the presenttechnology for treating the cancer and/or mammalian tissueoverexpressing PSMA or an effective amount of any embodiment of themodified antibody, modified antibody fragment, or modified bindingpeptide of the present technology for treating the cancer and/ormammalian tissue overexpressing PSMA or an effective amount of anyembodiment of the tripartite compound of the present technology fortreating the cancer and/or mammalian tissue overexpressing PSMA. In anrelated aspect, a method of treating a subject is provided, wherein themethod includes administering a targeting compound of the presenttechnology to the subject or administering a modified antibody, modifiedantibody fragment, or modified binding peptide of the present technologyto the subject. In any embodiment disclosed herein, it may be that thesubject suffers from cancer and/or mammalian tissue overexpressingprostate specific membrane antigen (“PSMA”). In any embodiment herein,it may be the administering includes administering an effective amountof any embodiment of the compounds of the present technology fortreating the cancer and/or mammalian tissue overexpressing PSMA of thecompound or an effective amount of any embodiment of the modifiedantibody, modified antibody fragment, or modified binding peptide of thepresent technology for treating the cancer and/or mammalian tissueoverexpressing PSMA or an effective amount of any embodiment of thetripartite compound of the present technology for treating the cancerand/or mammalian tissue overexpressing PSMA. The subject may suffer froma mammalian tissue expressing a somatostatin receptor, a bombesinreceptor, seprase, or a combination of any two or more thereof and/ormammalian tissue overexpressing PSMA. The mammalian tissue of anyembodiment disclosed herein may include one or more of a growth hormoneproducing tumor, a neuroendocrine tumor, a pituitary tumor, a vasoactiveintestinal peptide-secreting tumor, a small cell carcinoma of the lung,gastric cancer tissue, pancreatic cancer tissue, a neuroblastoma, and ametastatic cancer. In any embodiment disclosed herein, the subject maysuffer from one or more of a glioma, a breast cancer, an adrenalcortical cancer, a cervical carcinoma, a vulvar carcinoma, anendometrial carcinoma, a primary ovarian carcinoma, a metastatic ovariancarcinoma, a non-small cell lung cancer, a small cell lung cancer, abladder cancer, a colon cancer, a primary gastric adenocarcinoma, aprimary colorectal adenocarcinoma, a renal cell carcinoma, and aprostate cancer. In any embodiment disclosed herein, the composition(e.g., pharmaceutical composition) and/or medicament may be formulatedfor parenteral administration. In any embodiment disclosed herein, thecomposition (e.g., pharmaceutical composition) and/or medicament may beformulated for intravenous administration. In any embodiment disclosedherein, the administering step of the method may include parenteraladministration. In any embodiment disclosed herein, the administeringstep of the method may include intravenous administration.

In any of the above embodiments, the effective amount may be determinedin relation to a subject. “Effective amount” refers to the amount of acompound or composition required to produce a desired effect. Onenon-limiting example of an effective amount includes amounts or dosagesthat yield acceptable toxicity and bioavailability levels fortherapeutic (pharmaceutical) use including, but not limited to, thetreatment of e.g., one or more of a glioma, a breast cancer, an adrenalcortical cancer, a cervical carcinoma, a vulvar carcinoma, anendometrial carcinoma, a primary ovarian carcinoma, a metastatic ovariancarcinoma, a non-small cell lung cancer, a small cell lung cancer, abladder cancer, a colon cancer, a primary gastric adenocarcinoma, aprimary colorectal adenocarcinoma, a renal cell carcinoma, and aprostate cancer. Another example of an effective amount includes amountsor dosages that are capable of reducing symptoms associated with e.g.,one or more of a glioma, a breast cancer, an adrenal cortical cancer, acervical carcinoma, a vulvar carcinoma, an endometrial carcinoma, aprimary ovarian carcinoma, a metastatic ovarian carcinoma, a non-smallcell lung cancer, a small cell lung cancer, a bladder cancer, a coloncancer, a primary gastric adenocarcinoma, a primary colorectaladenocarcinoma, a renal cell carcinoma, and a prostate cancer, such as,for example, reduction in proliferation and/or metastasis of prostatecancer, breast cancer, or bladder cancer. The effective amount may befrom about 0.01 μg to about 1 mg of the compound per gram of thecomposition, and preferably from about 0.1 μg to about 500 μg of thecompound per gram of the composition. As used herein, a “subject” or“patient” is a mammal, such as a cat, dog, rodent or primate. Typicallythe subject is a human, and, preferably, a human suffering from orsuspected of suffering from one or more of a glioma, a breast cancer, anadrenal cortical cancer, a cervical carcinoma, a vulvar carcinoma, anendometrial carcinoma, a primary ovarian carcinoma, a metastatic ovariancarcinoma, a non-small cell lung cancer, a small cell lung cancer, abladder cancer, a colon cancer (such as colon adenocarcinoma), a primarygastric adenocarcinoma, a primary colorectal adenocarcinoma, a renalcell carcinoma, and a prostate cancer. The term “subject” and “patient”can be used interchangeably.

In any of the embodiments of the present technology described herein,the pharmaceutical composition may be packaged in unit dosage form. Theunit dosage form is effective in treating one or more of a glioma, abreast cancer, an adrenal cortical cancer, a cervical carcinoma, avulvar carcinoma, an endometrial carcinoma, a primary ovarian carcinoma,a metastatic ovarian carcinoma, a non-small cell lung cancer, a smallcell lung cancer, a bladder cancer, a colon cancer (such as colonadenocarcinoma), a primary gastric adenocarcinoma, a primary colorectaladenocarcinoma, a renal cell carcinoma, and a prostate cancer.Generally, a unit dosage including a compound of the present technologywill vary depending on patient considerations. Such considerationsinclude, for example, age, protocol, condition, sex, extent of disease,contraindications, concomitant therapies and the like. An exemplary unitdosage based on these considerations may also be adjusted or modified bya physician skilled in the art. For example, a unit dosage for a patientcomprising a compound of the present technology may vary from 1×10⁻⁴g/kg to 1 g/kg, preferably, 1×10⁻³ g/kg to 1.0 g/kg. Dosage of acompound of the present technology may also vary from 0.01 mg/kg to 100mg/kg or, preferably, from 0.1 mg/kg to 10 mg/kg. Suitable unit dosageforms, include, but are not limited to powders, tablets, pills,capsules, lozenges. suppositories. patches. nasal sprays, injectables,implantable sustained-release formulations, mucoadherent films, topicalvarnishes, lipid complexes, etc.

The pharmaceutical compositions may be prepared by mixing one or more ofthe compounds of Formulas I, IA, II, or any one of the modifiedantibodies, modified antibody fragments, or modified binding peptides ofthe present technology, or any embodiment of the tripartite compound ofthe present technology, pharmaceutically acceptable salts thereof,stereoisomers thereof, tautomers thereof, or solvates thereof, withpharmaceutically acceptable carriers, excipients, binders, diluents orthe like to prevent and treat disorders associated with cancer and/or amammalian tissue overexpressing PSMA. The compounds and compositionsdescribed herein may be used to prepare formulations and medicamentsthat treat e.g., prostate cancer, breast cancer, or bladder cancer. Suchcompositions may be in the form of, for example, granules, powders,tablets, capsules, syrup, suppositories, injections, emulsions, elixirs,suspensions or solutions. The instant compositions may be formulated forvarious routes of administration, for example, by oral, parenteral,topical, rectal, nasal, vaginal administration, or via implantedreservoir. Parenteral or systemic administration includes, but is notlimited to, subcutaneous, intravenous, intraperitoneal, andintramuscular, injections. The following dosage forms are given by wayof example and should not be construed as limiting the instant presenttechnology.

For oral, buccal, and sublingual administration, powders, suspensions,granules, tablets, pills, capsules, gelcaps, and caplets are acceptableas solid dosage forms. These can be prepared, for example, by mixing oneor more compounds of the instant present technology, or pharmaceuticallyacceptable salts or tautomers thereof, with at least one additive suchas a starch or other additive. Suitable additives are sucrose, lactose,cellulose sugar, mannitol, maltitol, dextran, starch, agar, alginates,chitins, chitosans, pectins, tragacanth gum, gum arabic, gelatins,collagens, casein, albumin, synthetic or semi-synthetic polymers orglycerides. Optionally, oral dosage forms can contain other ingredientsto aid in administration, such as an inactive diluent, or lubricantssuch as magnesium stearate, or preservatives such as paraben or sorbicacid, or anti-oxidants such as ascorbic acid, tocopherol or cysteine, adisintegrating agent, binders, thickeners, buffers, sweeteners,flavoring agents or perfuming agents. Tablets and pills may be furthertreated with suitable coating materials known in the art.

Liquid dosage forms for oral administration may be in the form ofpharmaceutically acceptable emulsions, syrups, elixirs, suspensions, andsolutions, which may contain an inactive diluent, such as water.Pharmaceutical formulations and medicaments may be prepared as liquidsuspensions or solutions using a sterile liquid, such as, but notlimited to, an oil, water, an alcohol, and combinations of these.Pharmaceutically suitable surfactants, suspending agents, emulsifyingagents, may be added for oral or parenteral administration.

As noted above, suspensions may include oils. Such oils include, but arenot limited to, peanut oil, sesame oil, cottonseed oil, corn oil andolive oil. Suspension preparation may also contain esters of fatty acidssuch as ethyl oleate, isopropyl myristate, fatty acid glycerides andacetylated fatty acid glycerides. Suspension formulations may includealcohols, such as, but not limited to, ethanol, isopropyl alcohol,hexadecyl alcohol, glycerol and propylene glycol. Ethers, such as butnot limited to, poly(ethyleneglycol), petroleum hydrocarbons such asmineral oil and petrolatum; and water may also be used in suspensionformulations.

Injectable dosage forms generally include aqueous suspensions or oilsuspensions which may be prepared using a suitable dispersant or wettingagent and a suspending agent. Injectable forms may be in solution phaseor in the form of a suspension, which is prepared with a solvent ordiluent. Acceptable solvents or vehicles include sterilized water,Ringer's solution, or an isotonic aqueous saline solution.Alternatively, sterile oils may be employed as solvents or suspendingagents. Typically, the oil or fatty acid is non-volatile, includingnatural or synthetic oils, fatty acids, mono-, di- or tri-glycerides.

For injection, the pharmaceutical formulation and/or medicament may be apowder suitable for reconstitution with an appropriate solution asdescribed above. Examples of these include, but are not limited to,freeze dried, rotary dried or spray dried powders, amorphous powders,granules, precipitates, or particulates. For injection, the formulationsmay optionally contain stabilizers, pH modifiers, surfactants,bioavailability modifiers and combinations of these.

Compounds of the present technology may be administered to the lungs byinhalation through the nose or mouth. Suitable pharmaceuticalformulations for inhalation include solutions, sprays, dry powders, oraerosols containing any appropriate solvents and optionally othercompounds such as, but not limited to, stabilizers, antimicrobialagents, antioxidants, pH modifiers, surfactants, bioavailabilitymodifiers and combinations of these. The carriers and stabilizers varywith the requirements of the particular compound, but typically includenonionic surfactants (Tweens, Pluronics, or polyethylene glycol),innocuous proteins like serum albumin, sorbitan esters, oleic acid,lecithin, amino acids such as glycine, buffers, salts, sugars or sugaralcohols. Aqueous and nonaqueous (e.g., in a fluorocarbon propellant)aerosols are typically used for delivery of compounds of the presenttechnology by inhalation.

Besides those representative dosage forms described above,pharmaceutically acceptable excipients and carriers are generally knownto those skilled in the art and are thus included in the instant presenttechnology. Such excipients and carriers are described, for example, in“Remingtons Pharmaceutical Sciences” Mack Pub. Co., New Jersey (1991),which is incorporated herein by reference. The instant compositions mayalso include, for example, micelles or liposomes, or some otherencapsulated form.

Specific dosages may be adjusted depending on conditions of disease, theage, body weight, general health conditions, sex, and diet of thesubject, dose intervals, administration routes, excretion rate, andcombinations of drugs. Any of the above dosage forms containingeffective amounts are well within the bounds of routine experimentationand therefore, well within the scope of the instant present technology.

Various assays and model systems can be readily employed to determinethe therapeutic effectiveness of the treatment according to the presenttechnology.

For the indicated condition, test subjects will exhibit a 10%, 20%, 30%,50% or greater reduction, up to a 75-90%, or 95% or greater, reduction,in one or more symptom(s) caused by, or associated with, the disorder inthe subject, compared to placebo-treated or other suitable controlsubjects.

In another aspect, the present technology provides a method of treatingcancer by administering an effective amount of the targeting compositionaccording to Formula II to a subject having cancer. Since a cancer celltargeting agent can be selected to target any of a wide variety ofcancers, the cancer considered herein for treatment is not limited. Thecancer can be essentially any type of cancer. For example, antibodies orpeptide vectors can be produced to target any of a wide variety ofcancers. The targeting compositions described herein are typicallyadministered by injection into the bloodstream, but other modes ofadministration, such as oral or topical administration, are alsoconsidered. In some embodiments, the targeting composition may beadministered locally, at the site where the target cells are present,i.e., in a specific tissue, organ, or fluid (e.g., blood, cerebrospinalfluid, etc.). Any cancer that can be targeted through the bloodstream isof particular consideration herein. Some examples of applicable bodyparts containing cancer cells include the breasts, lungs, stomach,intestines, prostate, ovaries, cervix, pancreas, kidney, liver, skin,lymphs, bones, bladder, uterus, colon, rectum, and brain. The cancer canalso include the presence of one or more carcinomas, sarcomas,lymphomas, blastomas, or teratomas (germ cell tumors). The cancer mayalso be a form of leukemia. In some embodiments, the cancer is a triplenegative breast cancer.

As is well known in the art, the dosage of the active ingredient(s)generally depends on the disorder or condition being treated, the extentof the disorder or condition, the method of administration, size of thepatient, and potential side effects. In different embodiments, dependingon these and other factors, a suitable dosage of the targetingcomposition may be precisely, at least, above, up to, or less than, forexample, 1 mg, 10 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1200 mg, or 1500 mg, or a dosagewithin a range bounded by any of the foregoing exemplary dosages.Furthermore, the composition can be administered in the indicated amountby any suitable schedule, e.g., once, twice, or three times a day or onalternate days for a total treatment time of one, two, three, four, orfive days, or one, two, three, or four weeks, or one, two, three, four,five, or six months, or within a time frame therebetween. Alternatively,or in addition, the composition can be administered until a desiredchange in the disorder or condition is realized, or when a preventativeeffect is believed to be provided.

The examples herein are provided to illustrate advantages of the presenttechnology and to further assist a person of ordinary skill in the artwith preparing or using the compounds of the present technology orsalts, pharmaceutical compositions, derivatives, prodrugs, or tautomericforms thereof. The examples herein are also presented in order to morefully illustrate the preferred aspects of the present technology. Theexamples should in no way be construed as limiting the scope of thepresent technology, as defined by the appended claims. The examples caninclude or incorporate any of the variations, aspects or embodiments ofthe present technology described above. The variations, aspects orembodiments described above may also further each include or incorporatethe variations of any or all other variations, aspects or embodiments ofthe present technology.

Examples

Exemplary Synthetic Procedures and Characterization

Materials and Instrumentation. All solvents and reagents, unlessotherwise noted, were purchased from commercial sources and used asreceived without further purification. Solvents noted as “dry” wereobtained following storage over 3 Å molecular sieves. Metal salts werepurchased from Strem Chemicals (Newburyport, Mass.) and were of thehighest purity available; Lu(ClO₄)₃ was provided as an aqueous solutioncontaining 15.1 wt % Lu. The bifunctional ligand p-SCN-Bn-DOTA waspurchased from Macrocyclics (Plano, Tex.). NMe₄OH was purchased as a 25wt % solution in H₂O (trace metals basis, Beantown Chemical, Hudson,N.H.). Hydrochloric acid (BDH Aristar Plus, VWR, Radnor, Pa.) and nitricacid (Optima, ThermoFisher Scientific, Waltham, Mass.) were of tracemetals grade. Both Chelex 100 (sodium form, 50-100 mesh) and human serumused for ²²⁵Ac-complex challenge assays were purchased from SigmaAldrich (St. Louis, Mo.). Deionized water (≥18 MΩ cm) was prepared onsite using either Millipore Direct-Q® 3UV or Elga Purelab Flex 2 waterpurification systems.

Reactions were monitored by thin-layer chromatography (TLC, WhatmanUV254 aluminum-backed silica gel). The HPLC system used for analysis andpurification of compounds consisted of a CBM-20A communications busmodule, an LC-20AP (preparative) or LC-20AT (analytical) pump, and anSPD-20AV UV/Vis detector monitoring at 270 nm (Shimadzu, Japan).Analytical chromatography was carried out using an Ultra Aqueous C18column, 100 Å, 5 μm, 250 mm×4.6 mm (Restek, Bellefonte, Pa.) at a flowrate of 1.0 mL/min, unless otherwise noted. Purification was performedwith an Epic Polar preparative column, 120 Å, 10 μm, 25 cm×20 mm (ESIndustries, West Berlin, N.J.) at a flow rate of 14 mL/min, unlessotherwise noted. Gradient HPLC methods were employed using a binarymobile phase that contained H₂O (A) and either MeOH (B) or ACN (C). HPLCMethod A: 10% B (0-5 min), 10-100% B (5-25 min). Method B: 10% C (0-5min), 10-100% C (5-25 min). Method C: 10% C (0-5 min), 10-100% C (5-40min). Method D: 10% C (0-5 min), 10-100% C (5-20 min). The solventsystems contained 0.1% trifluoroacetic acid (TFA), except for Method C,in which 0.2% TFA was used. NMR spectra were recorded at ambienttemperature on Varian Inova 300 MHz, 400 MHz, 500 MHz or 600 MHzspectrometers, or on a Bruker AV III HD 500 MHz spectrometer equippedwith a broadband Prodigy cryoprobe. Chemical shifts are reported in ppm.¹H and ¹³C NMR spectra were referenced to the TMS internal standard (0ppm), to the residual solvent peak, or to an acetonitrile internalstandard (2.06 ppm in D₂O spectra). ¹⁹F NMR spectra were referenced to amonofluorobenzene internal standard (−113.15 ppm). The splitting ofproton resonances in the reported ¹H spectra is defined as: s=singlet,d=doublet, t=triplet, q=quartet, m=multiplet, dt=doublet of triplets,td=triplet of doublets, and br=broad. IR spectroscopy was performed on aKBr pellet of sample using a Nicolet Avatar 370 DTGS (ThermoFisherScientific, Waltham, Mass.). High-resolution mass spectra (HRMS) wererecorded on an Exactive Orbitrap mass spectrometer in positive ESI mode(ThermoFisher Scientific, Waltham, Mass.). UV/visible spectra wererecorded on a Cary 8454 UV-Vis (Agilent Technologies, Santa Clara,Calif.) using 1-cm quartz cuvettes, unless otherwise noted. Elementalanalysis (EA) was performed by Atlantic Microlab, Inc. (Norcross, Ga.).

Synthesis and Characterization of Macropa Complexes, Macropa-NCS, andMacropa-NHC(S)NHCH₃.N,N′-bis[(6-carboxy-2-pyridil)methyl]-4,13-diaza-18-crown-6(H₂macropa.2HCl 4H₂O)^([102,103]) was prepared using1,7,10,16-tetraoxa-4,13-diazacyclooctadecane (7) that was eitherpurchased from EMD Millipore (Darmstadt, Germany) or synthesized vialiterature protocols.^([104]) Chelidamic acid monohydrate (1) waspurchased from TCI America (Portland, Oreg.). Dimethyl4-chloropyridine-2,6-dicarboxylate (2),^([105]) dimethyl4-azidopyridine-2,6-dicarboxylate (3),^([106]) and6-chloromethylpyridine-2-carboxylic acid methyl ester (8),^([102]) wereprepared via the indicated literature protocols.

Preparation of [La(macropa)]²⁺

To a suspension of H₂macropa.2HCl 4.H₂O (0.0233 g, 0.034 mmol) in2-propanol (0.6 mL) was added triethylamine (20 μL, 0.143 mmol). Thepale-gold solution was heated at reflux for 25 min before a solution ofLa(ClO₄)₃.6H₂O (0.0209 g, 0.038 mmol) in 2-propanol (0.5 mL) was addeddropwise. A precipitate formed immediately. The cream suspension wasstirred at reflux for an additional 1.5 h before it was cooled andcentrifuged. The supernatant was removed, and the pellet was washed with2-propanol (2-1 mL) and then air-dried on filter paper to give the titlecomplex as a pale-tan solid (0.0177 g) containing 0.64 equiv of2-propanol. ¹H NMR (500 MHz, D₂O, pD≈9) δ=7.87 (t, J=7.8 Hz, 2H), 7.54(d, J=7.8 Hz, 2H), 7.39 (d, J=7.6 Hz, 2H), 5.21 (d, J=15.7 Hz, 2H), 4.44(t, J=11.6 Hz, 2H), 4.09 (t, J=11.2 Hz, 4H), 4.01 (t, J=10.4 Hz, 2H),3.74 (d, J=9.9 Hz, 2H), 3.65-3.60 (m, 4H), 3.58-3.47 (m, 4H), 3.44 (d,J=10.8 Hz, 2H), 2.75 (td, J=13.1, 2.7 Hz, 2H), 2.56 (d, J=13.9 Hz, 2H),2.38 (d, J=14.0 Hz, 2H). ¹³C{¹H} APT NMR (126 MHz, D₂O, pD≈9) δ=172.62,158.70, 150.19, 140.94, 126.89, 122.32, 71.88, 70.12, 69.20, 68.05,60.14, 56.08, 54.01. EA Found: C, 35.16; H, 4.73; N, 5.91. Calc. forC₂₆H₃₅LaN₄O₈.2ClO₄.2H₂O.0.64iPrOH: C, 35.53; H, 4.71; N, 5.94. IR(cm⁻¹): 3443, 2913, 1630, 1596, 1461, 1370, 1265, 1083, 948, 839, 770,678, 617, 513. HPLC t_(R)=18.104 min (Method A). HRMS (m/z): 669.14289,335.07519; Calc for [C₂₆H₃₄LaN₄O₈]⁺ and [C₂₆H₃₅LaN₄O₈]²⁺, respectively:669.14346, 335.07537.

Preparation of [Lu(macropa)]⁺

To a suspension H₂macropa.2HCl.4H₂O (0.0730 g, 0.108 mmol) in 2-propanol(2 mL) was added triethylamine (61.5 μL, 0.441 mmol). The pale-goldsolution was heated at reflux for 25 min before a solution of aq.Lu(ClO₄)₃ (0.1372 g, 0.118 mmol Lu) in 2-propanol (1.8 mL) was addeddropwise. A precipitate formed immediately. After stirring at reflux oran additional 1 h, the cream suspension was triturated at RT for 20 hand then centrifuged. The supernatant was removed, and the pellet waswashed with 2-propanol (2×2 mL) and then air-dried on filter paper togive the title complex as a pale-tan solid (0.0605 g) containingresidual 2-propanol and triethylamine salt. ¹H NMR (600 MHz, D₂O,pD≈7-8) δ=7.85 (t, J=7.7 Hz, 2H), 7.52 (d, J=7.8 Hz, 2H), 7.37 (d, J=7.6Hz, 2H), 4.68 (d, J=16.3 Hz, 2H), 4.56 (td, J=11.2, 1.7 Hz, 2H),4.42-4.38 (m, 2H), 4.23-4.19 (m, 6H), 4.07 (d, J=16.3 Hz, 2H), 3.96-3.87(m, 2H), 3.71-3.63 (m, 4H), 3.38 (td, J=10.0, 4.7 Hz, 2H), 3.00 (m, 2H),2.93 (d, J=13.1 Hz, 2H), 2.52 (dt, J=14.8, 4.5 Hz, 2H). ¹³C{¹H} APT NMR(126 MHz, D₂O, pD≈7-8) δ=172.13, 158.67, 148.98, 141.81, 127.38, 122.83,75.33, 73.12, 71.97, 71.70, 64.65, 57.37, 55.08. IR (cm⁻¹): 3400, 1639,1396, 1274, 1091, 913, 770, 678, 622. HPLC t_(R)=not stable (Method A).HRMS (m/z). 705.17772; Calc for [C₂₆H₃₄LuN₄O₈]⁺: 705.17788.

Preparation of dimethyl 4-aminopyridine-2,6-dicarboxylate (4)

Dimethyl 4-azidopyridine-2,6-dicarboxylate (3, 0.9445 g, 4.0 mmol), 10%Pd/C (0.1419 g), and DCM:MeOH (1:1, 18 mL) were combined in around-bottom flask. After purging the flask with a balloon of H₂, thereaction was stirred vigorously at room temperature under an H₂atmosphere for 46 h. The gray mixture was diluted with DMF (450 mL) andfiltered through a bed of Celite. Following a subsequent filtrationthrough a 0.22 μm nylon membrane, the filtrate was concentrated at 60°C. under reduced pressure and further dried in vacuo to obtain 4 as apale-tan solid (0.824 g, 98% yield). ¹H NMR (500 MHz, DMSO-d₆): δ=7.36(s, 2H), 6.72 (s, 2H), 3.84 (s, 6H). ¹³C{H} APT NMR (126 MHz, DMSO-d6):δ=165.51, 156.24, 148.05, 111.99, 52.29. IR (cm⁻¹): 3409, 3339, 3230,1726, 1639, 1591, 1443, 1265, 996, 939, 787, 630, 543. HPLC t_(R)=9.369min (Method B). HRMS (m/z): 211.07213 [M+H]⁺; Calc: 211.07133.

Preparation of Ethyl 4-amino-6-(hydroxymethyl)picolinate (5)

To a refluxing suspension of 4 (0.677 g, 3.22 mmol) in absolute EtOH (27mL) was added NaBH₄ (0.1745 g, 4.61 mmol) portionwise over 1 h to give apale-yellow suspension. The reaction was then quenched with acetone (32mL) and concentrated at 60° C. under reduced pressure to a tan solid.The crude product was dissolved in H₂O (60 mL) and washed with ethylacetate (4×150 mL). The combined organics were dried over sodium sulfateand concentrated at 40° C. under reduced pressure. Further drying invacuo yielded 5 as a pale-yellow solid (0.310 g, 49% yield). ¹H NMR (300MHz, DMSO-d₆): δ=7.07 (d, 0.1=2.1 Hz, 1H), 6.78 (m, 1H), 6.32 (s, 2H),5.30 (t, J=5.8 Hz, 1H), 4.39 (d, J=5.6 Hz, 2H), 4.26 (q, J=7.1 Hz, 2H),1.28 (t, J=7.1 Hz, 3H). ¹³C APT NMR (126 MHz, DMSO-d₆) δ=165.57, 162.38,155.68, 147.25, 108.50, 107.01, 63.95, 60.61, 14.24. IR (cm⁻¹): 3439,3217, 2974, 2917, 1717, 1643, 1600, 1465, 1396, 1378, 1239, 1135, 1022,974, 865, 783. HPLC t_(R)=8.461 min (Method B). HRMS (m/z): 197.09288[M+H]⁺; Calc: 197.09207.

Preparation of Ethyl 4-amino-6-(chloromethyl)picolinate (6)

A mixture of thionyl chloride (2.5 mL) and 5 (0.301 g, 1.53 mmol) wasstirred in an ice bath for 1 h, and then at RT for 30 min. Theyellow-orange emulsion was concentrated at 40° C. under reduced pressureto an oily residue. The residue was neutralized with sat. aq. NaHCO₃ (12mL) and then extracted with ethyl acetate (75 mL). The organic extractwas washed with H₂O (2 mL), dried over sodium sulfate, and concentratedat 40° C. under reduced pressure. Further drying in vacuo gave 6 as anamber wax (0.287 g, 80% yield, corrected for residual ethyl acetate). ¹HNMR (500 MHz, DMSO-d₆) δ=7.18 (d, 1=2.1 Hz, 1H), 6.78 (d, 1=2.1 Hz, 1H),6.62 (br s, 2H), 4.62 (s, 2H), 4.29 (q, J=7.1 Hz, 2H), 1.30 (t, J=7.1Hz, 3H). ¹³C{¹H} APT NMR (126 MHz, DMSO-d₆) δ=164.75, 156.42, 156.19,147.17, 109.79, 109.50, 60.97, 46.47, 14.15. IR (cm⁻¹): 3452, 3322,3209, 2978, 2922, 1726, 1639, 1604, 1513, 1465, 1378, 1248, 1126, 1026,983, 861, 783, 752, 700. HPLC t_(R)=12.364 min (Method B). HRMS (m/z):215.05903 [M+H]⁺; Calc: 215.05818.

Preparation of Methyl6-((1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate(9.2TFA.1H₂O)

To a clear and colorless solution of1,7,10,16-tetraoxa-4,13-diazacyclooctadecane (7, 1.9688 g, 7.5 mmol) anddiisopropylethylamine (0.8354 g, 6.5 mmol) in dry ACN (1.075 L) at 75°C. was added dropwise a solution of 6 (0.9255 g, 5.0 mmol) in dry ACN(125 mL) over 2 h 40 min. The flask was then equipped with a condenserand drying tube, and the slightly-yellow solution was heated at refluxfor 42 h. Subsequently, the dark-gold solution containing fine, whiteprecipitate was concentrated at 60° C. under reduced pressure to anamber oil. To the crude oil was added 10% MeOH/H₂O containing 0.1% TFA(10 mL). The slight suspension was filtered, and the filtrate waspurified by preparative HPLC (Method A). Pure fractions were combined,concentrated at 60° C. under reduced pressure, and then lyophilized togive 9 (1.6350 g, 50% yield) as a pale-orange solid.

¹H NMR (500 MHz, DMSO-d₆) δ=8.75 (br s, 2H), 8.17-8.06 (m, 2H), 7.83(dd, J=7.4, 1.5 Hz, 1H), 4.68 (br s, 2H), 3.91 (s, 3H), 3.85 (br t,J=5.1 Hz, 4H), 3.69 (t, J=5.1 Hz, 4H), 3.59 (br s, 8H), 3.50 (br s, 4H),3.23 (br t, J=5.1 Hz, 4H). ¹³C{¹H} APT NMR (126 MHz, DMSO-d₆) δ 164.68,158.78-157.98 (q, TFA), 151.44, 147.13, 139.01, 128.63, 124.87,120.08-113.01 (q, TFA), 69.33, 69.00, 65.31, 64.60, 56.43, 53.29, 52.67,46.32. ¹⁹F NMR (470 MHz, DMSO-d₆) δ=−73.84. EA Found: C, 43.88; H, 5.29;N, 6.28. Calc. for C₂₀H₃₃N₃O₆.2CF₃COOH.1H₂O: C, 43.84; H, 5.67; N, 6.39.HPLC t_(R)=12.372 min (Method B). HRMS (m/z): 412.24568 [M+H]⁺; Calc:412.24421.

Preparation of Ethyl4-amino-6-((16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate(10)

Into a round-bottom flask equipped with a condenser and drying tube wereadded 9 (0.4210 g, 0.64 mmol), Na₂CO₃ (0.3400 g, 3.2 mmol), and dry ACN(10 mL). The pale-yellow suspension was heated to reflux over 15 min,after which 6 (0.1508 g, 0.70 mmol, corrected for residual ethylacetate) was added as a slight suspension in dry ACN (3.5 mL). Themixture was heated at reflux for 44 h and then filtered. The orangefiltrate was concentrated at 60° C. under reduced pressure to anorange-brown oil (0.612 g), which was used in the next step withoutfurther purification. HRMS (m/z): 590.32021 [M+H]⁺; Calc: 590.31844.

Preparation of4-Amino-6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinicacid (11.4TFA)

Compound 10 (0.612 g) was dissolved in 6 M HCl (7 mL) and heated at 90°C. for 17 h. The orange-brown solution containing slight precipitate wasconcentrated at 60° C. under reduced pressure to a pale-tan solid. Tothis solid was added 10% MeOH/H₂O containing 0.1% TFA (3 mL). The slightsuspension was filtered and the filtrate was purified by preparativeHPLC using Method A. Pure fractions were combined, concentrated at 60°C. under reduced pressure, and then lyophilized to give 11 as anoff-white solid (0.2974 g, 46% yield over 2 steps). ¹H NMR (500 MHz,DMSO-d₆) δ=8.13-8.08 (m, 2H), 7.80 (dd, J=7.3, 1.6 Hz, 1H), 7.64 (br s),7.24 (d, J=2.3 Hz, 1H), 6.76 (d, J=2.3 Hz, 1H), 4.74 (s, 2H), 4.15 (s,2H), 3.85 (t, J=5.0 Hz, 4H), 3.63 (t, J=5.1 Hz, 4H), 3.57-3.50 (m, 12H),3.09 (br t, J=5.2 Hz, 4H). ¹³C{¹H} NMR (126 MHz, DMSO-d₆) δ 165.96,163.37, 159.47, 158.78-157.98 (q, TFA), 151.93, 151.64, 148.25, 144.68,139.59, 128.43, 124.96, 120.79-113.68 (q, TFA), 109.40, 108.96, 70.03,69.89, 67.09, 65.16, 57.28, 55.85, 54.47, 53.81. ¹⁹F NMR (470 MHz,DMSO-d₆) δ=−74.03. EA Found: C, 40.60; H, 4.29; N, 7.04. Calc. forC₂₆H₃₇N₅O₈.4CF₃COOH: C, 40.69; H, 4.12; N, 6.98. IR (cm⁻¹): 3387, 3161,1735, 1670, 1204, 1130, 791, 722. HPLC t_(R)=11.974 min (Method B);11.546 min (Method D). HRMS (m/z): 548.26883 [M+H]⁺; Calc: 548.27149.

Preparation of6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)-4-isothiocyanatopicolinicacid (12, macropa-NCS)

A white suspension of 11 (0.1598 g, 0.16 mmol) and Na₂CO₃ (0.2540 g, 2.4mmol) was heated at reflux in acetone (10 mL) for 30 min before the slowaddition of CSCl₂ (305 μL of CSCl₂, 85%, Acros Organics). The resultingorange suspension was heated at reflux for 3 h and then concentrated at30° C. under reduced pressure to a pale-orange solid. The solid wasdissolved portionwise in 10% ACN/H₂O containing 0.2% TFA (8 mL total),filtered, and immediately purified by preparative HPLC using MethodC.^([108]) Pure fractions were combined, concentrated at RT underreduced pressure to remove the organic solvent, and then lyophilized.Fractions that were not able to be concentrated immediately were frozenat −80° C. Isothiocyanate 12 was obtained as a mixture of white andpale-yellow solid (0.0547 g) and was stored at −80° C. in ajar ofDrierite. Calculations from ¹H NMR and ¹⁹F NMR spectra of a sample of 12spiked with a known concentration of fluorobenzene estimated that 12 wasisolated as a tetra-TFA salt. ¹H NMR (400 MHz, DMSO-d₆) δ=8.17-8.06 (m,2H), 8.00 (s w/fine splitting, 1H), 7.84 (d, J=1.5 Hz, 1H), 7.81-7.75 (dw/fine splitting, J=7.16 Hz, 1H), 4.71 (s, 2H), 4.64 (s, 2H), 3.89-3.79(m, 8H), 3.62-3.46 (m, 16H). ¹⁹F NMR (470 MHz, DMSO-d₆) δ=−74.17. IR(cm⁻¹): ˜3500-2800, 2083, 2026, 1735, 1670, 1591, 1448, 1183, 1130, 796,717. HPLC t_(R)=15.053 min (Method B); 13.885 min (Method D). HRMS(m/z): 590.22600 [M+H]; Calc: 590.22791.

Preparation of6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)-4-(3-methylthioureido)picolinicacid (13, macropa-NHC(S)NHCH₃)

Compound 12 was prepared as described above using 0.0873 g (0.087 mmol)of 11, except the purification step was omitted. Instead, directly tothe crude solid was added 2 M methylamine in THF (4 mL). The tan-orangesuspension was stirred at RT for 2 h and then concentrated at RT underreduced pressure to a pale-peach solid. The solid was dissolved in 10%ACN/H₂O containing 0.2% TFA (2 mL), filtered, and purified bypreparative HPLC using Method C. Pure fractions were combined,concentrated at 50° C. under reduced pressure to remove the organicsolvent, and then lyophilized. The dark-gold, slightly sticky solid wasthen recrystallized from ACN with Et₂O. The suspension was centrifuged,and the pellet was washed with Et₂O (2×1.5 mL) and dried in vacuo togive 13 as a tan powder (0.0166 g, 22% unoptimized yield from 11). ¹HNMR (600 MHz, DMSO-d₆) δ=10.56 (s, 1H), 8.64 (br s, 1H), 8.26 (s, 1H),8.16 (s, 1H), 8.13-8.02 (m, 2H), 7.81-7.73 (d, J=7.40 Hz, 1H), 4.74-4.48(m, 4H), 3.82 (br s, 8H), 3.57 (br s, 8H), 3.54-3.25 (m, 8H), 2.97 (d,J=4.4 Hz, 4H). ¹³C{¹H} NMR (126 MHz, DMSO-d₆) δ 180.71, 165.44, 165.39,158.77-157.95 (q, TFA), 151.04, 150.96, 149.79, 147.95, 147.71, 139.22,127.76, 124.55, 119.68-112.66 (q, TFA), 116.45, 114.85, 69.36, 64.52,64.50, 57.00, 56.75, 53.42, 53.37, 31.02. ¹⁹F NMR (470 MHz, DMSO-d₆)δ=−74.49. EA Found: C, 44.66; H, 5.36; N, 9.83. Calc. forC₂₈H₄₀N₆O₈S.2CF₃COOH.1H₂O: C, 44.34, H, 5.12; N, 9.70. HPLC t_(R)=14.067min (Method B). HRMS (m/z): 621.26799 [M+H]⁺; Calc: 621.27011.

Preparation of Macropa-(OCH_(z)CH₂)-Ph-NCS

A schematic overview of the synthesis of an alternative embodiment ofMacropa-NCS, having improved stability is provided in FIG. 3. Thiscompound is evaluated as described below, and useful in the chelation ofradionuclides for their conjunction to antibodies, antibody fragments(e.g., antigen-binding fragments), and peptides, and their consequentuse in the manufacture of therapeutic compounds and targeted delivery oftherapeutic radiation. The detailed synthesis information is providedbelow.

A solution of compound 1 (0.725 g, 3 mmol), Ph₃P (0.802 g, 3.1 mmol) inCH₂Cl₂ (15 mL) was cooled to 0° C. under N₂. NBS (2.180.545 g, 3.3 mmol)was added portion wise for 5 min. The resulting solution was stirred for2 hrs at 0° C. and concentrated. Resulting crude product wasconcentrated and purified by combi-flash (5-10% EtOAc in hexane) to givecompound 2 (yield=76%).

To a solution of dimethyl 4-hydroxypyridine-2,6-dicarboxylate (0.253 g,1.2 mmol) and Cs₂CO₃ (0.650 g, 2 mmol) in DMF (6 mL) was added drop-wisecompound 2 (0.299 g, 1 mmol) in DMF (2 mL) under a N₂ condition. Theresulting solution was stirred for 24 hrs at room temperature. The DMFwas removed under reduced pressure and water was added, extracted withDCM. Resulting crude product was concentrated and purified bycombi-flash (5-10% EtOAc in hexane) to give compound 3 (yield=21%).

Compound 3 (0.215 g, 0.5 mmol) was dissolved in DCM: MeOH (2:1, 15 mL)and NaBH₄ (0.020 g, 0.6 mmol) was added in one portion at roomtemperature (under a N₂ condition). The resulting solution was stirredat same temperature for 3 hrs. The solvents were removed and water wasadded to the resulting residue and extracted into EtOAc. The organiclayer was removed under reduced pressure and resulting crude product waspurified by combi-flash (50-100% EtOAc in hexane) to give compound 4(yield=37%). A solution of compound 4 (0.201 g, 0.5 mmol), CBr₄ (0.198g, 0.6 mmol) and K₂CO₃ (0.103 g, 0.75 mmol) in CH₂Cl₂ (25 mL) was cooledto 0° C. (under N₂) was added drop-wise a solution of PPh₃ (0.157 g, 0.6mmol) in (DCM, 5 mL) for 10 min. The resulting reaction mixture wasstirred for 12 hrs at room temperature. Solvent was removed to result ina crude reaction mixture, which was purified by combi-flash (EtOAc inhexane) to give compound 5 (yield=70%).

To a clear and colorless solution of1,7,10,16-tetraoxa-4,13-diazacyclooctadecane (1.9688 g, 7.5 mmol) anddiisopropylethylamine (0.8354 g, 6.5 mmol) in dry ACN (1.075 L) at 75°C. was added dropwise a solution of methyl 6-(chloromethyl)picolinate(0.9255 g, 5.0 mmol) in dry ACN (125 mL) over 2 h 40 min. The flask wasthen equipped with a condenser and drying tube, and the slightly-yellowsolution was heated at reflux for 42 h. Subsequently, the dark-goldsolution containing fine, white precipitate was concentrated at 60° C.under reduced pressure to an amber gummy solid, compound 6, which wasused in the next step of the synthesis without any further purification.

To a stirred solution of compound 6 (0.205 g, 0.5 mmol) anddiisopropylethylamine (0.129 g, 1 mmol) in dry ACN (10 mL) was addedcompound 5 (0.233 g g, 0.5 mmol) in dry ACN (2 mL). The resulting ionsolution was stirred at r.t for 12 h. Solvent was removed and the crudecompound was purified by combi-flash using MeOH in DCM to yield compound7.

Compound 7 (0.08 g, 0.1 mmol) was dissolved in aq 6M HCl (5 mL) andstirred at room temperature for 2 h-3 h. After completion of thestarting material (evidenced by LCMS), aq HCl was removed under reducedpressure and the crude reaction mixture, containing compound 8 was usedin the next step of the synthesis without any further purification.

The crude deboc product was dissolved in THF:1M LiOH (1:1, 5 mL) andstirred until completion of the reaction. The resulting crude productwas purified by prep-HPLC giving compound 9.

NEt3 (7.6 mg, 0.076 mmol) was added to a solution of compound 9 (26 mg,0.038 mmol) in (8:2) acetonitrile and water (1 mL). Next, di-2-pyridylthionocarbonate (18 m g, 0.076 mmol) was added at room temperature andstirred vigorously for 1 h. The crude reaction solution was directlypurified by HPLC giving compound 10 (macropa-(OCH₂CH₂)-Ph-NCS).

X-Ray Diffraction Studies. Single crystals of H₂macropa.2HCl 4H₂Osuitable for x-ray diffraction were grown from a saturated H₂O:acetone(1:5) solution upon standing at room temperature. Single crystals of[La(Hmacropa)(H₂O)].(ClO₄)₂ were grown via vapor diffusion of THF intoan aqueous solution made acidic (pH ˜2) upon addition of the complex.Single crystals of [Lu(macropa)].ClO₄.DMF were grown via vapor diffusionof Et₂O into a DMF solution of the complex.

X-ray diffraction data for H₂macropa.2HCl.4H₂O,[La(Hmacropa)(H₂O)].(ClO₄)₂, and [Lu(macropa)].ClO₄.DMF were collectedon a Bruker APEX 2 CCD Kappa diffractometer (Mo Kα, λ=0.71073 Å) at 223K. The structures were solved through intrinsic phasing usingSHELXT^([109]) and refined against F² on all data by full-matrix leastsquares with SHELXL^([110]) following established refinementstrategies.^([111]) All non-hydrogen atoms were refined anisotropically.Hydrogen atoms were included in the model at geometrically calculatedpositions and refined using a riding model. Hydrogen atoms bound tonitrogen and oxygen were located in the difference Fourier synthesis andsubsequently refined semi-freely with the help of distance restraints.The isotropic displacement parameters of all hydrogen atoms were fixedto 1.2 times the U value of the atoms they are linked to (1.5 times formethyl groups). For [La(Hmacropa)(H₂O)].(ClO₄)₂, a partially occupiedsolvent molecule of water was included in the unit cell but could not besatisfactorily modeled. Therefore, that solvent was treated as a diffusecontribution to the overall scattering without using specific atompositions by the solvent masking function in Olex2.^([112])

La³⁺ and Lu³⁺ Titrations with Macropa. The pH of a 10 mM3-(N-morpholino)propanesulfonic acid (MOPS) buffer was adjusted to 7.4using aqueous NMe₄OH. The ionic strength was set at 100 mM using NMe₄Cl.Stock solutions of LaCl₃.6.8H₂O (40 mM) and LuCl₃.6H₂O (21 mM) wereprepared in 1 mM HCl. A stock solution of H₂macropa.2HCl.4H₂O (8.8 mM)was prepared in MOPS buffer. From these stock solutions, titrationsolutions containing macropa (100 μM) and either LaCl₃ or LuCl₃ wereprepared in MOPS. Each metal ion titration was carried out at RT byadding 5-10 μL aliquots of titrant to a cuvette containing 3000 μL ofmacropa (100 μM) in MOPS. Each sample was allowed to equilibrate for 5min following every addition before a spectrum was acquired.Complexation of the metal ion was monitored by the decrease inabsorbance at 268 nm, the λ_(max) of macropa. Titrant was added until nofurther spectral changes were detected.

Kinetic Inertness of La³⁺ and Lu³⁺ Complexes of Macropa: TranschelationChallenge. A stock solution of ethylenediaminetetraacetic acid (EDTA,100 mM) was made in MOPS buffer (prepared as described above) byadjusting the pH of the initial suspension to 6.6 using aqueous NMe₄OH.A stock solution of diethylenetriaminepentaacetic acid (DTPA, 125 mM)was prepared in H₂O by adjusting the pH to 7.4 as described for EDTA.This solution was serially diluted with H₂O to yield 12.5 mM and 1.25 mMsolutions of DTPA.

The preformed La³⁺ and Lu³⁺ complexes of macropa were challenged withEDTA. Challenges were initiated by adding an aliquot of solutioncontaining EDTA (98.7 mM) and macropa (100 μM) in MOPS buffer to eachsolution of complex. The final ratios of M:macropa:EDTA wereapproximately 1:1:20 (La) and 1:1:10 (Lu). Solutions were repeatedlyanalyzed by UV spectroscopy over the course of 21 days for any spectralchanges. The final pH of each solution was between 7.18 and 7.25.

The complex formed in situ between La³⁺ and macropa was more rigorouslychallenged with excess DTPA. A solution containing 500 μM of complex,prepared using the LaCl₃ and macropa stock solutions described above,was left to equilibrate for 5 min. Subsequently, it was portioned intocuvettes and diluted with either 125 mM DTPA, 12.5 mM DTPA, 1.25 mMDTPA, or MOPS to yield solutions containing 1000-, 100-, 10-, or 0-foldexcess DTPA and 100 μM concentration of macropa. These solutions wererepeatedly analyzed by UV spectroscopy over the course of 21 days forany spectral changes. The final pH of each solution was between 7.11 and7.42.

²²⁵Ac Radiolabeling of Macropa and DOTA. ²²⁵Ac and ²²⁵Ra were producedby the spallation of uranium carbide, separated downstream from otherradionuclides by a mass separator using the Isotope Separator andAccelerator (ISAC) isotope separation on-line (ISOL) facility at TRIUMF(Vancouver, BC, Canada), and were collected via literatureprotocols.^([103,104]) ²²⁵Ac was then separated from ²²⁵Ra via DGAcolumn^([105,105]) (branched, 50-100 μm, Eichrom Technologies LLC) andobtained in 0.05 M HNO₃ for use in radiolabeling experiments.Aluminum-backed TLC plates (silica gel 60, F₂₅₄, EMD Millipore,Darmstadt, Germany) were used to analyze 225Ac radiolabeling reactionprogress. Instant thin layer chromatography paper impregnated withsilica gel (iTLC-SG, Agilent Technologies, Mississauga, ON, Canada) wasused in La³⁺ and serum stability challenges. TLC plates were developedand then counted on a BioScan System 200 imaging scanner equipped with aBioScan Autochanger 1000 and WinScan software at least 8 h later toallow time for daughter isotopes to decay completely, ensuring that theradioactive signal measured was generated by parent ²²⁵Ac. Quantitativeradioactivity measurements of ²²⁵Ac, ²²¹Fr, and ²¹³Bi were determinedvia gamma-spectroscopy using a high-purity germanium (HPGe) detector(Canberra GR1520, Meriden, Conn.) calibrated using a NIST-traceablemixed ¹³³Ba and ¹⁵²Eu source. Detector dead time was maintained below10% for all measurements. Data was analyzed using Genie 2000 software(v3.4, Canberra, Meriden, Conn.).

Concentration Dependence. Various concentrations of macropa and DOTAwere radiolabeled with ²²⁵Ac³⁺ to determine the lowest concentration atwhich >95% radiolabeling still occurred. Stock solutions ofH₂macropa.2HCl.4H₂O (10⁻³-10⁻⁸ M) and H₄DOTA (10⁻³, 10⁻⁵, and 10⁻⁷M)were prepared in H₂O. For each radiolabeling reaction, ligand (10 μL)and 225Ac (10-26 kBq, 10-30 μL) were sequentially added to NH₄OAc buffer(pH 6, 0.15 M, 150 μL) to give final ligand concentrations of5.3×10⁻⁵-5.9×10⁻¹⁰ M for macropa and 5.9×10⁻⁵-5.9×10⁻⁹ M for DOTA. Thefinal pH of all labeling reactions was between 5.5 and 6. The reactionsolutions were maintained at ambient temperature or 80° C. Reactionprogress was monitored at 5 and 30 min by spotting 3-5 μL of thereaction solution onto TLC plates. The plates were developed with amobile phase of 0.4 M sodium citrate (pH 4) containing 10% MeOH and thencounted. Under these conditions, [²²⁵Ac(macropa)]⁺ and [²²⁵Ac(DOTA)]⁻remained at the baseline (R_(F)=0) and any unchelated ²²⁵Ac(²²⁵Ac-citrate) migrated with the solvent front (R_(F)=1). Radiochemicalyields (RCYs) were calculated by integrating area under the peaks on theradiochromatogram and dividing the counts associated with the²²⁵Ac-complex (R_(F)=0) by the total counts integrated along the lengthof the TLC plate.

Kinetic Inertness of ²²⁵Ac Complexes of Macropa and DOTA.

General. Stock solutions of La(NO₃)₃ (0.001 M or 0.1 M) were prepared inH₂O. To the radiolabeled samples containing macropa (10 μL of 10⁻⁵ Mstock; 1.0×10⁻¹⁰ moles) or DOTA (10 L of 10⁻³ M stock; 1.0×10⁻⁸ moles)and ²²⁵Ac (10 μL, 26 kBq) in NH₄OAc buffer (pH 6, 0.15 M, 150 μL), a50-fold mole excess of La³⁺ was added (5 μL, of 0.001 M or 0.1 M stockwere added to solutions containing macropa and DOTA, respectively). Thesolutions were kept at room temperature and analyzed by iTLC at severaltime points over the course of 8 days. The iTLC plates were developedusing citric acid (0.05 M, pH 5) as the eluent. Under these conditions,[²²⁵Ac(macropa)]⁺ and [²²⁵Ac(DOTA)]⁻ remained at the baseline (R_(F)=0)and any unchelated ²²⁵Ac (²²⁵Ac-citrate) migrated with the solvent front(R_(F)=1). Percent of complex remaining intact was calculated byintegrating area under the peaks on the radiochromatogram and dividingthe counts associated with the ²²⁵Ac-complex (R_(F)=0) by the totalcounts integrated along the length of the iTLC plate.

Transmetalation by La³⁺. [²²⁵Ac(macropa)]⁺ and [²²⁵Ac(DOTA)]⁻ wereprepared using 10⁻⁵ M and 10⁻³ M stock solutions (10 μL) of macropa andDOTA, respectively, to give final ligand concentrations of 5.9×10⁻⁷ M(macropa) and 5.9×10⁻⁵ M (DOTA). After confirming a radiochemical yieldof >90% by TLC using 0.4 M sodium citrate (pH 4) containing 10% MeOH asthe mobile phase, 160 μL of human serum (an equal volume based onlabeling reaction volume) were added to each radiolabeled solution. Acontrol solution was also prepared in which water was substituted forligand. The solutions were monitored over the course of 8 days by iTLC.The plates were developed with EDTA (50 mM, pH 5) as the eluent. Underthese conditions, [²²⁵Ac(macropa)]⁺ and [²²⁵Ac(DOTA)]⁻ complexesremained at the baseline (R_(F)=0) and any ²²⁵Ac (²²⁵Ac-EDTA) that hadbeen transchelated by serum migrated with the solvent front (R_(F)=1).Percent of complex remaining intact was calculated.

In Vivo Biodistribution of ²²⁵Ac Complexes of Macropa and DOTA. Allexperiments were approved by the Institutional Animal Care Committee(IACC) of the University of British Columbia and were performed inaccordance with the Canadian Council on Animal Care Guidelines. A totalof 9 female C57BL/6 mice (6-8 weeks old, 20-25 g) were used for thebiodistribution study of each radiometal complex, n=3 for each timepoint.

Macropa (100 μL of a 1 mg/mL solution in NH₄OAc) was diluted with 387 μLof NH₄OAc (1 M, pH 7), and an aliquot (203 μL) of ²²⁵Ac(NO₃)₃ (˜157 kBq)was then added; the pH of this solution was adjusted to 6.5-7 by theaddition of 1 M NaOH (210 μL, trace metal grade). After 5 min at ambienttemperature, the reaction solution was analyzed by TLC (0.4 M pH 4sodium citrate as the eluent), which confirmed >95% radiochemical yield.The reaction was allowed to proceed overnight, and the radiochemicalyield was again confirmed to be >95% the following morning. At thistime, mice were anesthetized by 2% isoflurane, and approximately 100 μL(10-15 kBq) of the [²²⁵Ac(macropa)]⁺ complex were injected into the tailvein of each mouse. After injection, mice were allowed to recover androam freely in their cages, and were euthanized by CO₂ inhalation at 15min, 1 h, or 5 h (n=3 at each time point) post-injection. Blood wascollected by cardiac puncture and placed into an appropriate test tubefor scintillation counting. Tissues collected included heart, liver,kidneys, lungs, small intestine, large intestine, brain, bladder,spleen, stomach, pancreas, bone, thyroid, tail, urine, and feces.Tissues were weighed and then counted with a calibrated gamma counter(Packard, Cobra II model 5002) using three energy windows: 60-120 keV(window A), 180-260 keV (window B), and 400-480 keV (window C). Countingwas performed both immediately after sacrifice and after 7 days; countswere decay corrected from the time of injection and then converted tothe percentage of injected dose (% ID) per gram of tissue (% ID/g). Nodifferences were noted between the data; therefore, the biodistributionsare reported using the data acquired immediately using window A.

The biodistribution studies of [²²⁵Ac(DOTA)] and ²²⁵Ac(NO₃)₃ werecarried out as described above for [²²⁵Ac(macropa)]⁺, with the followingmodifications. [²²⁵Ac(DOTA)⁻ was prepared by adding ²²⁵Ac(NO₃)₃ (338 μL,1.1 MBq) to a solution of DOTA (100 μg, 20 mg/mL in H₂O) in NH₄OAc (467μL, 0.15 M, pH 7). The pH of the solution was adjusted to 7 using NH₄OAc(150 μL, 1 M, pH 7) and the solution was heated at 85° C. for 45 min.RCY>99% was confirmed by TLC as described above. [²²⁵Ac(DOTA)]⁻ wasdiluted with saline to a final concentration of 0.05 MBq/100 μL, and 100μL were injected into each mouse. ²²⁵Ac(NO₃)₃ (˜58 μL, 0.4 MBq) wasdiluted and injected in the same manner as [²²⁵Ac(DOTA)]⁻. One mousethat was to be euthanized at the 5 h time point in the [²²⁵Ac(DOTA)]⁻study died shortly after injection. In the same manner, one mouse thatwas to be euthanized at the 1 h time point in the ²²⁵Ac(NO₃)₃ studydied.

Hydrolysis of Macropa-NCS and p-SCN-Bn-DOTA. To screw-capped vialscontaining approximately 1 mg of macropa-NCS (compound 12, n=4) orp-SCN-Bn-DOTA (n=5) was added 1 mL of 0.1 M pH 9.1 NaHCO₃ buffercontaining 0.154 M NaCl, which had been passed through a column ofpre-equilibrated Chelex. After stirring for 1 min, each solution wasfiltered through a 0.2 μm PES or PTFE membrane. Five μL aliquots wereremoved from the vials at various time points over the course of 46-72 hand analyzed by HPLC. Method D was employed for macropa-NCS. Method Bwas employed for p-SCN-Bn-DOTA using an Epic Polar C18 column, 120 Å, 10μm, 25 cm×4.6 mm (ES Industries, West Berlin, N.J.) at a flow rate of 1mL/min. Between samplings, the vials were stored at room temperature(23±1° C.) away from light. Hydrolysis was considered complete once thepeak at 13.8 min (corresponding to 12) or 18.417 min (correspondingtop-SCN-Bn-DOTA) had disappeared or had negligible integration. A linearregression performed on the plots of In peak area versus time providedthe pseudo-first order rate constant (k_(obs)) as the negative slope.The half-life (t_(1/2)) was calculated using the equationt_(1/2)=0.693/k_(obs). The half-life of each compound is reported as themean±1 standard deviation.

Titration of Macropa-NHC(S)NHCH₃ Conjugate with La³⁺. The titration ofthe macropa-NHC(S)NHCH₃ conjugate (13) with La³⁺ was carried out at pH7.4 for macropa, except that the stock solution of 13 (0.760 mM) wasprepared in ACN instead of MOPS. The amount of ACN in the sample did notexceed 3.3% by volume. A wait time of 3 min after the addition of eachaliquot was found to be sufficient for the sample to reach equilibriumbefore spectral acquisition. Complexation of the metal ion was monitoredusing the increase in absorbance at 300 nm. The pH of the solution atthe end of the titration was 7.43.

Kinetic Inertness of La-Macropa-NHC(S)NHCH₃: Transchelation Challenge.Solutions of diethylenetriaminepentaacetic acid (DTPA; 125 mM and 12.5mM) were prepared in MOPS buffer (pH 7.4). A MOPS solution containingmacropa-NHC(S)NHCH₃ (126.7 μM, 16.7% ACN by volume) and LaCl₃ (126.2 μM)was prepared using the stock solutions described above and was left toequilibrate for 10 min. Subsequently, it was portioned into cuvettes anddiluted with either 125 mM DTPA, 12.5 mM DTPA, or MOPS to yieldsolutions containing 1000-, 100-, or 0-fold excess DTPA. The finalconcentration of macropa-NHC(S)NHCH₃ in each cuvette was 25.3 μM. Thesesolutions were repeatedly analyzed by UV spectrophotometry over thecourse of 21 days for any spectral changes. The final pH of eachsolution was between 7.42 and 7.49. The experiment was performed intriplicate.

Exemplary Synthesis and Biological Activity of ²²⁵Ac-macropa-RPS-070

Preparation of Di-tert-butyl(((S)-1-(tert-butoxy)-6-(3-(3-ethynylphenyl)ureido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate(214)

Alkyne 214 was prepared according to published methods^([247]) andisolated as an off-white powder. ¹H NMR (500 MHz, CDCl₃) δ=7.90 (s, 1H),7.58 (t, 1H, J=1.7 Hz), 7.51 (dd, 1H, J₁=8.2 Hz, J₂=1.3 Hz), 7.18 (t,1H, J=7.9 Hz), 7.05 (d, 1H, J=7.7 Hz), 6.38 (d, 1H, J=7.9 Hz), 6.28 (brs, 1H), 5.77 (d, 1H, J=6.9 Hz), 4.32 (m, 1H), 4.02 (m, 1H), 3.53 (m,1H), 3.05 (m, 1H), 3.00 (s, 1H), 2.39 (m, 2H), 2.07 (m, 1H), 1.88 (m,1H), 1.74 (m, 1H), 1.62 (m, 1H), 1.49-1.37 (m, 4H), 1.41 (s, 18H), 1.37(s, 9H).

Preparation of 2,5-Dioxopyrrolidin-1-ylN²-(((9H-fluoren-9-yl)methoxy)carbonyl)-N⁶-(tert-butoxycarbonyl)-L-lysinate(215)

A suspension of Fmoc-L-Lys(Boc)-OH (5.0 g, 10.7 mmol) andN,N′-disuccinimidyl carbonate (2.74 g, 10.7 mmol) in CH₂Cl₂ (50 mL) wasstirred at room temperature under argon. Then DIPEA (1.86 mL, 10.7 mmol)was added, and the suspension was stirred overnight. The solvent wasevaporated under reduced pressure and the crude product was purified byflash chromatography (0-100% EtOAc in hexane). Lysine 215 was isolatedas a white powder (2.5 g, 41%). ¹H NMR (500 MHz, CDCl₃) δ=7.76 (d, 2H,J=7.6 Hz), 7.59 (d, 2H, J=7.3 Hz), 7.40 (t, 2H, J=7.4 Hz), 7.32 (t, 2H,J=7.3 Hz), 5.46 (br s, 1H), 4.71 (m, 2H), 4.45 (m, 2H), 4.23 (t, 1H,J=6.6 Hz), 3.14 (br s, 2H), 2.85 (s, 4H), 2.02 (m, 1H), 1.92 (m, 1H),1.58 (m, 4H), 1.44 (s, 9H).

Preparation of tert-ButylN²—(N²-(((9H-fluoren-9-yl)methoxy)carbonyl)-N⁶-(tert-butoxycarbonyl)-L-lysyl)-N⁶-((benzyloxy)carbonyl)-L-lysinate(216)

A suspension of L-Lys(Z)-OtBu.HCl (1.49 g, 4.0 mmol) in CH₂Cl₂ (15 mL)was treated with DIPEA (0.87 mL, 5.0 mmol). To the resulting mixture wasadded a solution of lysine 215 (2.2 g, 3.9 mmol) in CH₂Cl₂ (10 mL), andthe reaction was stirred overnight at room temperature under argon. Itwas then washed with saturated NaCl solution, and the organic layer wasdried over MgSO₄, filtered and concentrated under reduced pressure. Thecrude product was purified by flash chromatography (0-100% EtOAc inhexane), and di-lysine 216 was isolated as a white powder (2.2 g, 72%).¹H NMR (500 MHz, CDCl₃) δ=7.76 (d, 2H, J=7.5 Hz), 7.59 (d, 2H, J=7.3Hz), 7.40 (t, 2H, J=7.5 Hz), 7.32 (m, 8H), 6.69 (br s, 1H), 5.60 (br s,1H), 5.06 (m, 4H), 4.72 (br s, 1H), 4.43 (m, 1H), 4.38 (m, 1H), 4.21 (m,1H), 3.14 (m, 4H), 1.85 (m, 2H), 1.73 (m, 2H), 1.50 (m, 4H), 1.46 (s,9H), 1.44 (s, 9H), 1.39 (m, 4H).

Preparation of 2,5-Dioxopyrrolidin-1-yl 2-(4-iodophenyl)acetate (217)

A solution of 2-(4-iodophenyl)acetic acid (786 mg, 3.0 mmol) and EDC-HCl(671 mg, 3.5 mmol) in CH₂Cl₂ (20 mL) was stirred for 15 min at roomtemperature under argon. Then N-hydroxysuccinimide (368 mg, 3.2 mmol)and NEt3 (0.56 mL, 4.0 mmol) were added and the reaction was stirred for7 h. It was then washed with saturated NaCl solution, and the organiclayer was dried over MgSO₄, filtered and concentrated under reducedpressure. The crude residue was purified by flash chromatography (0-100%EtOAc in hexane), and the NHS ester 217 was isolated as a white solid(760 mg, 70%). ¹H NMR (500 MHz, CDCl₃) δ=7.69 (d, 2H, J=7.9 Hz), 7.09(d, 2H, J=7.9 Hz), 3.88 (s, 2H), 2.83 (s, 4H).

Preparation of tert-ButylN²—(N²-(1-azido-3,6,9,12,15,18-hexaoxahenicosan-21-oyl)-N⁶-(tert-butoxycarbonyl)-L-Iysyl)-N⁶-((benzyloxy)carbonyl)-L-lysinate(218)

To a solution of Fmoc-protected di-lysine 216 (768 mg, 0.97 mmol) inCH₂Cl₂ (4 mL) was added NHEt₂ (2.07 mL, 20 mmol). The solution wasstirred overnight at room temperature. The solvents were removed underreduced pressure, and the crude product, a yellow oil, was used withoutfurther purification. To a solution of this oil (183 mg, 0.32 mmol) inCH₂Cl₂ (3 mL) were added successively solutions of NEt3 (57 μL, 0.41mmol) in CH₂Cl₂ (1 mL) and azido-PEG₆-NHS ester (100 mg, 0.21 mmol;Broadpharm, USA) in CH₂Cl₂ (1 mL), and the reaction was stirredovernight at room temperature. It was then diluted with CH₂Cl₂ andwashed successively with H₂O and saturated NaCl solution. The organiclayer was dried over MgSO₄, filtered and concentrated under reducedpressure to give azide 218 as a colorless oil (184 mg; 95%) without needfor further purification. Mass (ESI+): 926.4 [M+H]⁺. Calc. Mass=925.54.

Preparation of Di-tert-butyl(((S)-1-(tert-butoxy)-6-(3-(3-(1-((9S,12S)-9-(tert-butoxycarbonyl)-12-(4-((tert-butoxycarbonyl)amino)butyl)-3,11,14-trioxo-1-phenyl-2,17,20,23,26,29,32-heptaoxa-4,10,13-triazatetratriacontan-34-yl)-1H-1,2,3-triazol-4-yl)phenyl)ureido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate(219)

A solution of 100 μL of 0.5 M CuSO₄ and 100 μL of 1.5 M sodium ascorbatein DMF (0.5 mL) was mixed for 5 min and was then added to a solution of218 (184 mg, 0.20 mmol) and 214 (132 mg, 0.21 mmol) in DMF (2.5 mL). Theresulting mixture was stirred at room temperature for 45 min. It wasthen concentrated under reduced pressure and the crude residue waspurified by flash chromatography (0-30% MeOH in EtOAc) to give triazole219 as an orange oil (285 mg; 87%). Mass (ESI+): 1557.2 [M+H]⁺. Calc.Mass=1555.90.

Preparation of Di-tert-butyl(((S)-1-(tert-butoxy)-6-(3-(3-(1-((23S,26S)-26-(tert-butoxycarbonyl)-23-(4-((tert-butoxycarbonyl)amino)butyl)-33-(4-iodophenyl)-21,24,32-trioxo-3,6,9,12,15,18-hexaoxa-22,25,31-triazatritriacontyl)-1H-1,2,3-triazol-4-yl)phenyl)ureido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate(220)

Cbz-Protected triazole 219 (285 mg, 0.18 mmol) was dissolved in MeOH (15mL) in a two-neck flask. To the solution was added 10% Pd/C (20 mg), andthe suspension was shaken and the flask evacuated. The suspension wasthen placed under an H₂ atmosphere and stirred overnight. It wasfiltered through celite, and the filter cake was washed three times withMeOH. The combined filtrate was concentrated under reduced pressure togive the free amine as a colorless oil (117 mg; 45%) that was usedwithout further purification. Mass (ESI+): 1423.8 [M+H]⁺. Calc.Mass=1422.77. To a solution of the amine (117 mg, 82 μmol) in CH₂Cl₂ (4mL) was added a solution of DIPEA (23 μL, 131 mmol) in CH₂Cl₂ (1 mL),and the mixture was stirred at room temperature under argon. Then asolution of 217 (37 mg, 103 μmol) in CH₂Cl₂ (2 mL) was added, and thereaction was stirred at room temperature for 2 h. It was then pouredinto H₂O (10 mL) and the layers were separated. The organic layer wasdried over MgSO₄, filtered and concentrated under reduced pressure togive the crude product as a colorless semi-solid. The crude product waspurified by prep TLC (10% MeOH in EtOAc) to give phenyl iodide 220 as acolorless oil (34 mg; 25%). Mass (ESI+): 1666.6 [M+H]⁺. Calc.Mass=1665.80.

Preparation of(((S)-1-Carboxy-5-(3-(3-(1-((23S,26S)-26-carboxy-23-(4-(3-(2-carboxy-6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)pyridin-4-yl)thioureido)butyl)-33-(4-iodophenyl)-21,24,32-trioxo-3,6,9,12,15,18-hexaoxa-22,25,31-triazatritriacontyl)-1H-1,2,3-triazol-4-yl)phenyl)ureido)pentyl)carbamoyl)-L-glutamicacid (221, macropa-RPS-070)

To a solution of 220 (34 mg, 20 μmol) in CH₂Cl₂ (2 mL) was added TFA(0.5 mL), and the reaction was stirred at room temperature for 5 h. Itwas then concentrated under reduced pressure, and the crude product wasdiluted with H₂O and lyophilized to give the free amine as a TFA salt.Mass (ESI+): 1342.5 [M+H]⁺. Mass (ESI−): 1340.6 [M−H]⁻. Calc.Mass=1341.50. To a solution of the amine (9 mg, 6.7 μmol) in DMF (0.5mL) was added a solution of macropa-NCS (15 mg, 25.4 μmol) in DMF (0.5mL). Then DIPEA (300 μL, 1.72 mmol) was added and the reaction wasstirred at room temperature for 2 h. The volatiles were removed underreduced pressure and the crude product was purified by prep HPLC to givemacropa-RPS-070 (221) as a white powder (5.4 mg; 42%). Mass (ESI+):1932.76 [M+H]⁺. 1931.09 [M+H]⁻. Calc. Mass=1931.91.

Preparation of Radiosynthesis of ²²⁵Ac-macropa-RPS-070

General. All reagents were purchased from Sigma Aldrich unless otherwisenoted, and were reagent grade. Hydrochloric acid (HCl) was traceSELECT®(>99.999%) for trace analysis quality. Aluminum-backed silica thin layerchromatography (TLC) plates were purchased from Sigma Aldrich. Stocksolutions of 0.05 M HCl and 1 M NH₄OAc were prepared by dilution inMilli-Q® water.

Radiolabeling Procedure. To a solution of ²²⁵Ac(NO₃)₃ (Oak RidgeNational Laboratory, USA) in 0.05 M HCl (17.9 MBq in 970 μL) was added20 μL of a 1 mg/mL solution of macropa-RPS-070 in DMSO. The pH wasraised to 5-5.5 by addition of 90 μL 1 M NH₄OAc. The reaction wasallowed to stand at room temperature for 20 min with periodic shaking.Then, 200 μL of the reaction solution was removed and diluted with 3.8mL of normal saline (0.9% NaCl in deionized H₂O; VWR) to give a solutionwith a concentration of 910 kBq/mL. An aliquot was removed from thefinal solution and spotted onto an aluminum-backed silica TLC plate todetermine radiochemical yield. An aliquot of the ²²⁵Ac(NO₃)₃ solution in0.05M HCl was spotted in a parallel lane as a control. The plate wasimmediately run in a 10/6 v/v MeOH/10 mM EDTA mobile phase, and thenallowed to stand for 8 h to enable radiochemical equilibrium to bereached. The plate was visualized on a Cyclone Plus Storage PhosphorSystem (Perkin Elmer) following a 3-min exposure on the phosphor screen.The radiochemical yield was expressed as a ratio of²²⁵Ac-macropa-RPS-070 to total activity and was determined to be 98.1%.

Biodistribution Studies with ²²⁵Ac-macropa-RPS-070.

Cell Culture. The PSMA-expressing human prostate cancer cell line,LNCaP, was obtained from the American Type Culture Collection. Cellculture supplies were from Invitrogen unless otherwise noted. LNCaPcells were maintained in RPMI-1640 medium supplemented with 10% fetalbovine serum (Hyclone), 4 mM L-glutamine, 1 mM sodium pyruvate, 10 mMN-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 2.5 mg/mLD-glucose, and 50 μg/mL gentamicin in a humidified incubator at 37°C./5% CO₂. Cells were removed from flasks for passage or for transfer to12-well assay plates by incubating them with 0.25%trypsin/ethylenediaminetetraacetic acid (EDTA).

Inoculation of Mice with Xenografts. All animal studies were approved bythe Institutional Animal Care and Use Committee of Weill CornellMedicine and were undertaken in accordance with the guidelines set forthby the USPHS Policy on Humane Care and Use of Laboratory Animals.Animals were housed under standard conditions in approved facilitieswith 12 h light/dark cycles. Food and water was provided ad libitumthroughout the course of the studies. Hairless male nu/nu mice werepurchased from the Jackson Laboratory. For inoculation in mice, LNCaPcells were suspended at 4×10⁷ cells/mL in a 1:1 mixture of PBS:Matrigel(BD Biosciences). Each mouse was injected in the left flank with 0.25 mLof the cell suspension. Biodistributions were conducted when tumors werein the range 100-400 mm³.

Biodistribution of ²²⁵Ac-macropa-RPS-070 in LNCaP xenograft mice.Fifteen LNCaP xenograft tumor-bearing mice (5 per time point) wereinjected intravenously with a bolus injection of 85-95 kBq and 100 ng(50 μmol) of each ligand. The mice were sacrificed by cervicaldislocation at 4, 24 and 96 h post injection. A blood sample wasremoved, and a full biodistribution study was conducted on the followingorgans (with contents): heart, lungs, liver, small intestine, largeintestine, stomach, spleen, pancreas, kidneys, muscle, bone, and tumor.Tissues were weighed and counted on a 2470 Wizard Automatic GammaCounter (Perkin Elmer). 1% ID/mL samples were counted prior to andfollowing each set of tissue samples to enable decay correction to beundertaken. Counts were corrected for decay and for activity injected,and tissue uptake was expressed as percent injected dose per gram (%ID/g). Standard error measurement was calculated for each data point.

TABLE F Organ distribution of ²²⁵Ac-macropa-RPS-070 at t = 4 h, 24 h,and 96 h following intravenous injection in LNCaP xenograft mice (n = 5per time point). Values expressed as % ID/g. 1 2 3 4 5 Mean SEM 4 hBlood 0.90654 0.55246 1.11808 0.8276 0.65638 0.81221 0.0986 Heart0.75759 0.65317 0.77395 0.75148 0.6585 0.71894 0.02604 Lungs 0.995580.60669 1.25979 0.98587 0.88664 0.94691 0.10516 Liver 1.62187 1.346321.74207 1.68077 1.3957 1.55735 0.0788 Small 0.1998 0.16282 0.31040.24413 0.17094 0.21762 0.02721 intestine Large 1.36298 0.65162 1.274190.91656 0.81901 1.00487 0.13563 intestine Stomach 0.33963 0.2471 0.304170.4109 0.21221 0.3028 0.03489 Spleen 1.40902 0.70804 1.61264 1.108150.8756 1.14269 0.16632 Pancreas 0.55487 0.41637 0.55317 0.4675 0.66040.53047 0.04182 Kidneys 65.5884 20.5274 108.233 33.654 33.0707 52.214615.8618 Muscle 0.68006 0.80579 0.72817 0.67666 0.65617 0.70937 0.02684Bone 1.14861 1.12335 1.48731 0.92036 1.15463 1.16685 0.09106 Tumor6.73177 10.7309 23.8367 15.3682 7.50352 12.8342 3.1429 24 h Blood0.34825 0.31324 0.22083 0.29453 0.27697 0.29076 0.0211 Heart 0.522560.56334 0.4521 0.47914 0.46483 0.49639 0.02052 Lungs 0.53778 0.450770.46083 0.4286 0.44831 0.46526 0.01887 Liver 1.57844 1.47552 1.137761.14264 1.48473 1.36382 0.09305 Small 0.08784 0.09914 0.08822 0.094660.10376 0.09473 0.00309 intestine Large 0.13296 0.1259 0.13252 0.134250.13176 0.13148 0.00145 intestine Stomach 0.1296 0.12119 0.1119 0.146750.15329 0.13255 0.00773 Spleen 0.62075 0.65764 0.62013 0.57685 0.585540.61218 0.01443 Pancreas 0.39847 0.39119 0.50347 0.33315 0.31944 0.389140.03252 Kidneys 4.98792 4.25707 3.94586 3.66457 4.10348 4.19178 0.22185Muscle 0.61193 0.5149 0.44832 0.78028 0.44579 0.56025 0.06276 Bone1.27255 1.06645 0.83943 1.00576 0.69755 0.97635 0.09828 Tumor 11.61639.26927 7.50158 4.41446 8.04683 8.16969 1.17583 96 h Blood 0.190420.19188 0.15206 0.16528 0.23822 0.18757 0.01475 Heart 0.39939 0.423980.42861 0.45863 0.45595 0.43331 0.01098 Lungs 0.30165 0.50912 0.469440.37811 0.36979 0.40562 0.03717 Liver 0.79406 0.8144 0.73301 0.79170.79415 0.78546 0.01374 Small 0.04372 0.0577 0.03752 0.04431 0.041360.04492 0.00341 intestine Large 0.04349 0.09663 0.04522 0.04198 0.039270.05332 0.01087 intestine Stomach 0.03442 0.04708 0.03448 0.028450.02366 0.03362 0.00393 Spleen 0.48373 0.394 0.44261 0.43481 0.539660.45896 0.02469 Pancreas 0.09848 0.37696 0.30549 0.31625 0.33352 0.286140.04847 Kidneys 1.30286 1.3239 2.00405 1.39866 1.45955 1.4978 0.12958Muscle 0.3022 0.52492 0.25089 0.29815 0.2528 0.32579 0.05095 Bone0.86391 0.86874 0.83831 1.12223 0.82042 0.90272 0.05557 Tumor 4.042594.07799 6.73954 4.58107 4.84503 4.85724 0.49449

Conjugation of Macropa-NCS and p-SCN-Bn DOTA to Trastuzumab.

General. All glassware was washed overnight in 1M HCl. Saline (0.154 MNaCl) and all buffer solutions were passed through a column ofChelex-100 pre-equilibrated with the appropriate buffer. Trastuzumab(Tmab, Genentech) was purified using a Zeba spin desalting column (2 mLor 5 mL, 40 MWCO, Thermo Scientific, Waltham, Mass.) according to themanufacturer's protocol, with saline as the mobile phase. Theconcentration of purified Tmab was calculated via the Beer-Lambert lawusing A₂₈₀ and an 6280 of 1.446 mL mg⁻¹ cm⁻¹.^([107]) Purified Tmab andTmab conjugates were stored at 4° C.

Conjugation of Macropa-NCS to Tmab. A stock solution containing 4.4mg/mL of macropa-NCS (12) was prepared in 0.1 M pH 9.1 NaHCO₃ buffercontaining 0.154 M NaCl and was stored at −80° C. The stability of 12during storage was verified by analytical HPLC. To a portion of Tmab insaline (74 μL) were added 12 (52 μL) and NaHCO₃ buffer (266 μL), so thatthe final concentrations of Tmab and 12 were 5.1 mg/mL and 0.59 mg/mL,respectively. Macropa-NCS was estimated to be in 16-fold molar excess toTmab based on a molecular weight of 1045.76 g/mol for 12 (tetra-TFAsalt). The pH of this solution was between 8 and 9 by litmus paper. Thesolution was rocked gently at room temperature for 17.5 h and thenpurified using a spin column.

Conjugation of p-NCS-Bn-DOTA to Tmab. A stock solution containing 3.05mg/mL of p-NCS-Bn-DOTA was prepared in H₂O and stored at −80° C. To aportion of Tmab in saline (66 μL) were added p-NCS-Bn-DOTA (49 μL) andNaHCO₃ buffer (274.5 μL), so that the final concentrations of Tmab andp-NCS-Bn-DOTA were 5.1 mg/mL and 0.38 mg/mL (16-fold molar excess of L),respectively. The pH of this solution was between 8 and 9 by litmuspaper. The solution was rocked gently at room temperature for 17.5 h andthen purified using a spin column.

Determination of Conjugate Protein Concentration by BCA Assay. Theconcentration of protein in macropa-Tmab and DOTA-Tmab conjugates wasdetermined using the Pierce™ BCA Protein Assay kit (Thermo Scientific,Waltham, Mass., microplate protocol). Tmab was employed as the proteinstandard. A stock solution of purified Tmab was diluted with saline andthe concentration of this solution (1.83 mg/mL) was determined using aNanoDrop 1000 Spectrophotometer (Thermo Scientific, Waltham, Mass.). Thestandard curve was linear (r²=0.9966) over the concentration rangemeasured (0-1828 μg/mL). The protein concentration of each conjugate wascalculated from two independent dilutions, each measured in triplicate,and the results were averaged to give a protein concentration of 4.557mg/mL for macropa-Tmab and 2.839 mg/mL for DOTA-Tmab.

Ligand-to-Protein Ratio Analysis by MALDI-ToF. The average number ofmacropa or DOTA ligands conjugated to Tmab was determined by MALDI-ToFMS/MS on a Bruker autoflex speed at the Alberta Proteomics and MassSpectrometry Facility (University of Alberta, Canada) using a proceduredescribed elsewhere.^([108]) Purified Tmab and the conjugates wereanalyzed in duplicate, and the [M+H]⁺ mass signals from thechromatograms were averaged for each compound. The ligand-to-protein(L:P) ratio for each conjugate was obtained by subtracting the molecularweight of Tmab from the molecular weight of the conjugate, andsubsequently dividing by the mass of the bifunctional ligand.

²²⁵Ac Radiolabeling of Tmab Conjugates and Serum Stability of Complexes.

General. Instant thin layer chromatography paper impregnated with silicagel (iTLC-SG, Agilent Technologies, Mississauga, ON, Canada) was used tomonitor the progress of ²²⁵Ac radiolabeling reactions and to determineserum stability. TLC plates were developed as described below and thencounted on a BioScan System 200 imaging scanner equipped with a BioScanAutochanger 1000 and WinScan software at least 8 h later to allow timefor daughter isotopes to decay completely, ensuring that the radioactivesignal measured was generated by parent²²⁵Ac.

²²⁵Ac Radiolabeling Studies. In a total reaction volume of 200 μL madeup with NH₄OAc buffer (pH 6, 0.15 M), ²²⁵Ac (10 or 20 kBq, 7-10 μL) wasmixed with 25-100 μg of either macropa-Tmab (5.5-22 μL) or DOTA-Tmab(8.81-35.2 μL), and the pH was adjusted to ˜5 with NaOH. A controlsolution was also prepared in which unmodified Tmab (25 μg) wassubstituted in place of conjugate. The reaction solutions weremaintained at ambient temperature and analyzed at 5 min, 30 min, 1 h, 2h, 3 h, and 4 h by spotting 8 μL in triplicate on iTLC strips. Thestrips were developed with a mobile phase of 0.05 M citric acid (pH 5).Under these conditions, ²²⁵Ac-macropa-Tmab and ²²⁵Ac-DOTA-Tmab remainedat the baseline of the plate (R_(F)=0) and any unchelated ²²⁵Ac(²²⁵Ac-citrate) migrated with the solvent front (R_(F)=1). Radiochemicalyields (RCYs) were calculated by integrating area under the peaks on theradiochromatogram and dividing the counts associated with the²²⁵Ac-complex (R_(F)=0) by the total counts integrated along the lengthof the TLC plate.

Stability of ²²⁵Ac-macropa-Tmab in Human Serum. A solution of²²⁵Ac-macropa-Tmab was prepared using 100 μg of protein. Afterconfirmation by TLC that a RCY of >95% had been achieved, human serumwas thawed to room temperature and added to the radiolabeledimmunoconjugate to give a solution containing 90% serum by volume. Thesample was incubated at 37° C. At various time points over the course of7 days, aliquots (15-30 μL) were removed from the sample and spotted intriplicate onto iTLC strips. The strips were developed using an EDTA (50mM, pH 5.2) mobile phase and counted. Under these conditions,²²⁵Ac-macropa-Tmab remained at the baseline (R_(F)=0) and any ²²⁵Ac(²²⁵Ac-EDTA) that had been transchelated by serum migrated with thesolvent front (R_(F)=1). Percent of complex remaining intact wascalculated.

As an additional challenge, separate aliquots (39 μL) were also removedfrom the serum sample on days 1 and 7 and mixed with 50 mM DTPA (pH 7,13 μL) to challenge off any ²²⁵Ac that was only loosely bound by theradioimmunoconjugate. After incubation of this solution at 37° C. for 15minutes, an aliquot (30 μL) was spotted in triplicate on iTLC plates anddeveloped using an EDTA (50 mM, pH 5.2) mobile phase. Percent of complexremaining intact was calculated.

In Vivo Biodistribution Studies of [²²⁵Ac(macropa)]+, [²²⁵Ac(DOTA)]⁻,and ²²⁵Ac(NO₃)₃.

TABLE 1 Organ distribution of ²²⁵Ac complexes following intravenousinjection in mice. Adult C57BL/6 mice were injected with[²²⁵Ac(macropa)]⁺, [²²⁵Ac(DOTA)]⁻, or ²²⁵Ac(NO₃)₃ and sacrificed after15 min, 1 h, or 5 h. Values for each time point are given as % ID/g (n =3) using energy window A (60-120 keV). Organ 15 min SD 1 h SD 5 h SD[²²⁵Ac(macropa)]⁺ blood 5.11 2.82 0.40 0.38 0.01 0.01 urine 1378.82971.53 489.11 26.75 12.78 6.10 feces 0.91 1.18 0.28 0.14 3.46 1.06 heart2.19 0.60 0.31 0.24 0.10 0.11 liver 2.28 0.41 0.75 0.18 0.39 0.03kidneys 27.55 7.51 13.36 17.13 0.74 0.06 lungs 5.98 1.81 0.51 0.36 0.010.04 small 2.64 1.08 1.10 0.47 0.29 0.20 intestines large 2.40 0.52 0.360.10 0.49 0.22 intestines brain 0.26 0.09 0.12 0.07 0.02 0.02 bladder46.74 24.65 6.23 7.44 4.25 5.27 spleen 2.52 1.08 0.51 0.19 0.11 0.03stomach 2.97 0.72 0.41 0.08 0.01 0.06 pancreas 1.46 0.64 0.19 0.16 0.100.06 bone 2.52 0.34 0.31 0.16 0.05 0.10 (femur + joint) thyroids 28.2317.90 3.18 2.21 0.10 7.95 tail 8.84 1.56 1.82 1.11 0.14 0.09[²²⁵Ac(DOTA)]⁻ blood 5.2881 2.9807 0.1144 0.0203 0.0140 0.0024 urine1467.9186 1073.9229 158.6102 141.1945 1.1612 0.3653 feces 6.2730 8.72840.2035 0.2433 5.5318 1.7685 heart 2.3335 0.7337 0.1012 0.0853 0.06640.0091 liver 2.2520 0.5051 0.2715 0.1973 0.1010 0.0063 kidneys 27.65666.8974 1.4020 0.2124 0.6172 0.0168 lungs 5.7556 1.7234 0.1555 0.08000.0390 0.0135 small 2.6370 1.3350 1.7207 2.1165 0.0967 0.0232 intestineslarge 2.3348 0.7436 0.1229 0.0551 0.2026 0.1073 intestines brain 0.26550.0598 0.0224 0.0123 0.0213 0.0021 bladder 48.2703 26.4988 4.7351 4.96210.3551 0.0335 spleen 2.5905 1.3909 0.0938 0.0322 0.1380 0.0733 stomach2.7440 0.8312 0.1367 0.1078 0.0852 0.0100 pancreas 1.5090 0.6828 0.07430.0752 0.0677 0.0090 bone 2.6298 0.6802 0.4487 0.0586 0.2063 0.0231(femur + joint) thyroids −5.7725 27.0550 2.3564 2.7015 3.6425 1.8897tail 8.8606 1.1879 0.8091 0.1272 0.3057 0.0766 ²²⁵Ac(NO₃)₃ blood 40.9666.455 20.8234 0.8102 1.9886 0.5457 urine 5.527 3.460 4.5194 0.48034.8267 3.6549 feces 0.240 0.070 0.2189 0.1167 0.9445 0.7998 heart 8.5572.698 4.4261 1.2771 1.3450 0.2326 liver 22.899 1.788 39.8269 4.506259.8156 10.4928 kidneys 10.468 1.897 7.2170 1.5026 4.6910 2.3005 lungs12.757 2.883 8.2412 1.9189 4.1871 3.8011 small 2.002 0.094 1.5594 0.31911.3704 0.4345 intestines large 1.116 0.145 0.6035 0.4502 0.6479 0.2782intestines brain 0.614 0.283 0.2995 0.0893 0.0452 0.0343 bladder 1.4770.689 0.9047 0.0759 1.4947 2.4402 spleen 22.733 4.962 34.8831 1.676862.9614 12.7041 stomach 2.348 0.250 1.6211 0.0147 2.6131 1.4450 pancreas2.366 0.922 2.1771 0.8907 0.4874 0.4300 bone 2.764 0.757 2.4707 0.11983.5460 0.6374 (femur + joint) thyroids 4.391 1.511 2.5988 4.9499 −2.70522.9758 tail 7.459 5.674 5.7939 1.8506 23.4055 19.5704

TABLE 2 Organ distribution of ²²⁵Ac complexes following intravenousinjection in mice. Adult C57BL/6 mice were injected with[²²⁵Ac(macropa)]⁺, [²²⁵Ac(DOTA)]⁻, or ²²⁵Ac(NO₃)₃ and sacrificed after15 min, 1 h, or 5 h. Values for each time point are given as % ID/g (n =3) using energy window B (180-260 keV). Organ 15 min SD 1 h SD 5 h SD[²²⁵Ac(macropa)]⁺ blood 5.23 2.93 0.39 0.38 0.00 0.01 urine 1541.601105.98 517.19 11.65 13.51 6.04 feces 1.04 0.92 0.27 0.21 3.49 1.18heart 2.39 0.80 0.20 0.31 −0.04 0.12 liver 2.17 0.40 0.70 0.16 0.36 0.01kidneys 27.86 7.39 12.97 17.16 0.78 0.14 lungs 5.83 1.81 0.54 0.25 −0.050.14 small 2.59 1.19 0.94 0.46 0.29 0.21 intestines large 2.53 0.57 0.220.18 0.45 0.27 intestines brain 0.23 0.06 0.12 0.11 −0.01 0.04 bladder47.64 25.00 5.92 8.15 3.69 6.69 spleen 2.55 1.54 0.23 0.26 0.09 0.06stomach 3.29 1.03 0.33 0.26 0.04 0.14 pancreas 1.63 0.73 0.12 0.22 −0.120.16 bone 2.69 0.63 0.17 0.11 0.02 0.01 (femur + joint) thyroids −2.2212.06 0.10 5.33 −6.94 8.77 tail 9.39 1.59 1.82 1.04 0.13 0.05[²²⁵Ac(DOTA)]⁻ blood 5.6357 3.2852 0.1127 0.0403 0.0292 0.0172 urine1635.4394 1233.7980 159.1628 143.0187 3.6967 3.3377 feces 1.0222 0.98590.2349 0.2923 3.3534 1.0198 heart 2.7276 0.7955 0.1378 0.1197 0.08790.0591 liver 2.1817 0.4921 0.2672 0.1890 0.2712 0.2370 kidneys 28.08586.9019 1.2560 0.1319 0.6718 0.1380 lungs 6.0147 1.8416 0.1946 0.10770.1289 0.0320 small 2.5009 1.2567 1.8809 2.3424 0.2065 0.1617 intestineslarge 2.5365 0.7142 0.0813 0.0554 0.2527 0.1980 intestines brain 0.27350.1473 0.0248 0.0120 0.0513 0.0110 bladder 54.4696 32.7034 4.7141 5.10770.7521 0.0884 spleen 2.9076 1.5773 0.0825 0.0965 0.0834 0.2219 stomach2.7311 0.9322 0.1379 0.1390 0.1789 0.0565 pancreas 1.4929 1.2189 0.07460.0806 0.1266 0.0354 bone 3.0357 0.7199 0.4126 0.0368 0.1478 0.1689(femur + joint) thyroids 1.6601 7.1867 2.6514 6.1376 16.2357 11.0860tail 9.4746 1.5429 0.8973 0.0672 0.1634 0.0768 ²²⁵Ac(NO₃)₃ blood 41.56286.0720 21.4460 1.0862 2.0018 0.5989 urine 5.0951 2.4036 7.0564 2.09843.3142 2.6426 feces 0.3857 0.1799 0.3300 0.1741 1.0201 0.9002 heart8.3605 2.5149 4.5832 1.4669 1.3948 0.3318 liver 23.6091 2.1849 41.09955.1387 62.0765 10.0091 kidneys 9.6424 1.6131 6.8770 1.0099 3.8752 1.6179lungs 12.9714 2.7540 8.4426 1.9117 4.3379 3.9596 small 1.9641 0.18531.5192 0.2815 1.2201 0.3708 intestines large 1.1570 0.1960 0.5629 0.34600.6744 0.2893 intestines brain 0.6536 0.2639 0.3247 0.0633 0.0290 0.0219bladder 1.6996 0.7289 0.8092 0.2576 1.5234 2.6761 spleen 24.0497 5.353137.1540 0.1801 65.9117 13.1934 stomach 2.3704 0.3085 1.5867 0.28532.5322 1.4903 pancreas 2.2821 0.9761 2.1579 0.8408 0.4455 0.3936 bone2.7487 0.6608 2.7705 0.0730 3.8533 0.7991 (femur + joint) thyroids9.6295 8.0396 5.7426 3.0938 −4.6044 2.5708 tail 8.0722 6.2766 6.42012.1693 25.4744 20.7518

TABLE 3 Organ distribution of ²²⁵Ac complexes following intravenousinjection in mice. Adult C57BL/6 mice were injected with[²²⁵Ac(macropa)]⁺, [²²⁵Ac(DOTA)]⁻, or ²²⁵Ac(NO₃)₃ and sacrificed after15 min, 1 h, or 5 h. Values for each time point are given as % ID/g (n =3) using energy window C (400-480 keV). Organ 15 min SD 1 h SD 5 h SD[²²⁵Ac(macropa)]⁺ blood 6.49 4.64 0.54 0.55 0.04 0.03 urine 2387.661987.77 641.63 49.58 22.27 8.14 feces 1.26 2.00 0.69 0.50 5.27 2.17heart 2.87 1.51 0.23 0.97 0.28 0.84 liver 2.72 0.61 1.08 0.45 0.55 0.08kidneys 33.46 5.62 17.38 21.12 1.07 0.37 lungs 7.55 3.24 0.84 0.62 0.150.14 small 3.46 2.44 1.62 0.76 0.42 0.28 intestines large 3.02 1.11 0.790.51 0.68 0.17 intestines brain 0.17 0.10 0.23 0.13 −0.01 0.08 bladder64.68 45.85 9.00 3.35 8.52 10.72 spleen 3.79 2.96 0.48 1.92 0.43 0.14stomach 3.45 1.29 0.17 0.77 0.13 0.23 pancreas 3.00 2.21 0.43 1.01 0.130.29 bone 3.74 1.27 0.70 0.36 0.08 0.16 (femur + joint) thyroids −6.4666.56 8.34 11.63 19.89 30.96 tail 11.75 0.66 2.57 1.39 0.28 0.10[²²⁵Ac(DOTA)]⁻ blood 7.2941 4.1461 0.1102 0.0707 — — urine 2691.06151906.4694 177.6788 168.4716 — — feces 1.5693 1.8307 0.4091 0.4652 — —heart 2.5579 2.0110 0.2857 0.2702 — — liver 2.9046 0.8757 0.2841 0.2157— — kidneys 40.4489 10.8186 1.4787 0.7053 — — lungs 7.3872 1.9528 0.25510.1695 — — small 3.8916 2.4605 2.0201 2.4443 — — intestines large 3.84191.8882 0.1381 0.2122 — — intestines brain 0.1588 0.0692 0.0380 0.0968 —— bladder 76.0987 42.8592 6.9149 4.5152 — — spleen 1.5598 1.6847 0.22280.4642 — — stomach 3.2425 2.1465 0.1720 0.2911 — — pancreas 1.02901.1339 0.1730 0.1437 — — bone 4.4224 1.8431 0.5654 0.2432 — — (femur +joint) thyroids −109.5394 150.5455 3.5247 36.1530 — — tail 13.47313.2236 1.0280 0.3206 — — ²²⁵Ac(NO₃)₃ blood 42.3521 6.5376 11.373615.9719 2.1769 0.7500 urine 19.8282 14.9210 104.9103 130.5319 5.85488.2799 feces 0.4896 0.2884 0.1122 0.1587 0.8535 0.2061 heart 9.09923.1686 3.3464 4.3204 1.2018 0.1929 liver 24.1147 1.8809 23.6180 33.254554.1727 4.7696 kidneys 14.2266 4.1528 6.2070 7.2061 4.2061 1.5123 lungs14.4797 2.7960 5.2078 7.2810 5.4923 4.6341 small 2.0956 0.0803 3.55481.8035 1.2922 0.6032 intestines large 1.5716 0.8096 0.4366 — 1.02590.5032 intestines brain 0.6755 0.2338 0.4402 0.1057 0.0430 0.0773bladder 1.9351 2.1420 2.2929 1.3941 3.4975 5.8177 spleen 25.4263 6.001138.1082 — 62.2357 17.5694 stomach 2.4232 0.3667 2.3350 — 2.0358 1.6514pancreas 2.4405 0.5887 1.8508 — 0.4643 0.3109 bone 3.4560 0.9882 2.7213— 3.5851 1.4683 (femur + joint) thyroids 3.5934 1.5023 0.0000 — −0.44553.5100 tail 9.1381 7.4041 9.0877 — 28.4443 30.7841

In Vivo Studies of ²²⁵Ac-macropa-Tmab.

At the time points indicated in Table 4 below, an aliquot of complex inserum was removed and either directly analyzed by radio-TLC or firstmixed with excess DTPA to remove any loosely-bound ²²⁵Ac. Thedecay-corrected values shown represent % activity associated with thecomplex at R_(F)=0 on the TLC plate after exposure to an EDTA mobilephase. Reported uncertainties (±1 SD) were derived from spotting TLCplates in triplicate at each time point. The % intact complex remainingwas not significantly different for samples subjected to the DTPAchallenge versus those that were not (p>0.05, 2-tail t-test). Theresults demonstrate that ²²⁵Ac remains strongly bound by macropa-Tmab inhuman serum over a 7-day period.

TABLE 4 Complex stability (% intact complex remaining) of²²⁵Ac-macropa-Tmab in human serum at 37° C. 1 h 1 day 3 days 7 daysWithout DTPA Challenge 96.4 ± 0.9 99.0 ± 0.5 98.7 ± 0.6 99.2 ± 0.4 WithDTPA Challenge — 91.5 ± 12  — 97.1 ± 1.6

Characterization of Eighteen-Membered Macrocyclic Ligands for IonChelation

Radium-223 (²²³Ra) is the first therapeutic alpha (α)-emittingradionuclide to be approved for clinical use in cancer patients, and iseffective in eradicating bone metastases. To harness the therapeuticpotential of a-particles for soft-tissue metastases, the strategy oftargeted alpha-particle therapy (TAT) has emerged, whereby lethala-emitting radionuclides are conjugated to tumor-targeting vectors usingbifunctional chelators to selectively deliver cytotoxic alpha radiationto cancer cells. Actinium-225 (²²⁵Ac) was examined for use in TAT owingto its long 10-day half-life that is compatible with antibody-basedtargeting vectors and 4 high-energy α-emissions that are extremelylethal to cells. The 12-membered tetraaza macrocycle H₄DOTA is currentlythe state of the art for the chelation of the ²²⁵Ac³⁺ ion, however, thethermodynamic stabilities of complexes of H₄DOTA decrease as the ionicradius of the metal ion increases, indicating that this ligand is notoptimal for chelation of the of the Ac³⁺ ion (the largest +3 ion on theperiodic table). The macrocyclic complexes of the present technologyprovide a significant and unexpected improvement over known complexes,where the present examples (H₂macropa and H₂macropa-NCS; Scheme 1)illustrate the improved ²²⁵Ac bifunctional chelators according to thepresent technology.

Scheme 1. Structures of H₂macropa, H₂macropa-NCS (“macropa-NCS”), andmacropa-(OCH₂CH₂)-Ph-NCS.

Previous studies have shown that macropa, for which the thermodynamicaffinity for the whole lanthanide series was evaluated, is selective forthe larger metal ions La³⁺, Pb²⁺, and Am³⁺ over the smaller Lu³⁺, Ca²⁺,and Cm³⁺ ions.^([226]) Without wishing to be bound by theory it wasbelieved that macropa would effectively chelate the large Ac³⁺ ion.Before assessing its Ac-chelation properties, complex formation wasevaluated in situ between macropa and cold La³⁺ and Lu³⁺ ions. In thesestudies, La³⁺ was used as a non-radioactive surrogate for ²²⁵Ac³⁺because it is chemically similar albeit slightly smaller (1.03 Å, CN 6).Complexation of the smaller Lu³⁺ ion (0.861 Å, CN 6) by macropa wasinvestigated to probe its size-selectivity. La³⁺ and Lu³⁺ titrationsconfirmed the high affinity of these metal ions for macropa at pH 7.4,consistent with the previously measured stability constants (logK_(LaL)=14.99, log K_(LuL)=8.25).^([24]) The kinetic inertness of thesecomplexes formed in situ was investigated by challenging them with anexcess of either ethylenediaminetetraacetic acid (EDTA) ordiethylenetriaminepentaacetic acid (DTPA) chelators that have a higherthermodynamic affinity than macropa for Lu³⁺ and La³⁺ ions.^([27]) TheLu³⁺ ion was transchelated within 1 min upon the addition of only 10equiv of EDTA, whereas the La³⁺ complex remained intact for up to 21days in the presence of 1000 equiv of DTPA. These results demonstratethat, despite a strong thermodynamic preference for DTPA to transchelateLa³⁺, the high level of kinetic inertness of the macropa complexinhibits this process on a detectable time scale.

The La³⁺ and Lu³⁺ complexes of macropa were isolated and theirsolid-state structures were elucidated by X-ray crystallography (FIGS.1A-1D). The La³⁺ and Lu³⁺ ions reside above the 18-membered macrocycle,and the two picolinate arms are positioned on the same side of themacrocycle. The coordination sphere of the Lu³⁺ ion is satisfied by theten donors of macropa with both picolinate arms deprotonated; bycontrast, the larger La³⁺ ion forms an 11-coordinate complex by theincorporation of an inner-sphere water molecule that penetrates themacrocycle. The ability of macropa to form stable 11-coordinatecomplexes is of particular significance because recent EXAFS studieshave demonstrated that Ac³⁺ prefers a coordination number of 11 inaqueous solutions.^([29,30])

Macropa was examined for the chelation of the larger, radioactive²²⁵Ac³⁺ ion and compared to DOTA. Both ligands (59 μM) were incubatedwith ²²⁵Ac (26 kBq) in 0.15 M NH₄OAc buffer at pH 5.5-6, and thecomplexation reaction was monitored by radio-TLC after 5 min.Remarkably, macropa complexed all the ²²⁵Ac after merely 5 min at RT,whereas DOTA only complexed 10% under these conditions. At 100-foldlower concentration (0.59 μM) of macropa, a L:M ratio of only 1800,radiolabeling was still complete at RT in 5 min. At this concentration,DOTA failed to form a complex with ²²⁵Ac. Taken together, these studiesreveal macropa to exhibit excellent radiolabeling kinetics at ambienttemperature and submicromolar ligand concentration, conditions underwhich DOTA fails.

The long half-life of ²²⁵Ac necessitates its stable complex retention invivo to avoid off-target damage to normal tissues arising from therelease of free ²²⁵Ac³⁺. Furthermore, the stability of ²²⁵Ac complexesagainst transmetalation and transchelation needs to be high. Todetermine the kinetic inertness, [²²⁵Ac(macropa)]⁺ was challeneged withLa³⁺ because of the established high affinity of macropa for this metalion. A 50-fold excess of La³⁺ with respect to ligand concentration wasadded ²²⁵Ac-radiolabeled solutions of macropa (0.59 μM) at RT. Over 7days, 98% of the ²²⁵Ac complex remained intact by radio-TLC, signifyingthat a large molar equivalent of La³⁺ is unable to displace ²²⁵Ac³⁺. Thestability of [²²⁵Ac(macropa)]⁺ in human serum was also evaluated byradio-TLC and revealed that ²²⁵Ac³⁺ remains complexed by macropa for atleast 8 days.

Evaluation of the Biodistribution of [²²⁵Ac(macropa)]⁺ Complexes

The in vivo stability [²²⁵Ac(macropa)]⁺ was examined by comparing itsbiodistribution to those of ²²⁵Ac(NO₃)₃ and [²²⁵Ac(DOTA)]⁻. C57BL/6 micewere injected via tail vein with 10-50 kBq of each radiometal complexand were sacrificed after 15 min, 1 h, or 5 h. The amount of ²²⁵Acretained in each organ was quantified by gamma counting and reported asthe percent of injected dose per gram of tissue (% ID/g). The results ofthese studies are compiled in Tables 1-3. Inadequate stability of an²²⁵Ac complex leading to the loss of radioisotope in vivo is manifestedby the accumulation of ²²⁵Ac in the liver, spleen, and bone ofmice.^([11,12,32]) FIG. 2A demonstrates slow blood clearance andexcretion, coupled to large accumulation in the liver and spleen of theuncomplexed ²²⁵Ac(NO₃)₃. The biodistribution profile of[²²⁵Ac(macropa)]⁺ (FIG. 3B) differs markedly from that of ²²⁵Ac(NO₃)₃.[²²⁵Ac(macropa)]⁺ was rapidly cleared from mice, with very littleactivity measured in blood by 1 h post injection. Most of the injecteddose was renally excreted and subsequently detected in the urine,demonstrating the moderate kidney and bladder uptake of[²²⁵Ac(macropa)]⁺ observed in mice at 15 min and 1 h post injection. Ofsignificance, [²²⁵Ac(macropa)]⁺ did not accumulate in any organ over thetime course of the study, indicating that the complex does not releasefree ²²⁵Ac³⁺ in vivo. Its biodistribution profile was similar to that of[²²⁵Ac(DOTA)]⁻ (FIG. 3C), which has been previously shown to retain²²⁵Ac in vivo.^([7])

Synthesis and Characterization of [²²⁵Ac(macropa)]⁺ TAT Complexes

Due to the inherent stability of the [²²⁵Ac(macropa)]⁺ complexes,macropa was incorporated into tumor-targeting constructs. To facilitateits conjugation, a reactive isothiocyanate functional group wasinstalled onto one of the picolinate arms of macropa to give the novelbifunctional ligand macropa-NCS (Scheme 1). As illustrated in videsupra, macropa-NCS was synthesized over 8 steps and characterized byconventional techniques. For one tumor-targeting construct, macropa-NCSwas s conjugated to trastuzumab (Tmab), an FDA-approved monoclonalantibody that targets the human epidermal growth factor receptor 2(HER2) in breast and other cancers.^([33]) With a biological half-lifeof several weeks,^([34,35]) Tmab is an ideal vector to shuttle thelong-lived ²²⁵Ac radionuclide to tumor cells. ²²⁵Ac-macropa-Tmabdisplayed excellent stability in human serum at 37° C.; after 7days, >99% of the complex remained intact (Table 4). Together, theseresults highlight the efficacy of macropa as a chelator for ²²⁵Ac inantibody constructs as well as other cancer-targeted constructs.

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While certain embodiments have been illustrated and described, a personwith ordinary skill in the art, after reading the foregoingspecification, can effect changes, substitutions of equivalents andother types of alterations to the compounds of the present technology orsalts, pharmaceutical compositions, derivatives, prodrugs, metabolites,tautomers or racemic mixtures thereof as set forth herein. Each aspectand embodiment described above can also have included or incorporatedtherewith such variations or aspects as disclosed in regard to any orall of the other aspects and embodiments.

The present technology is also not to be limited in terms of theparticular aspects described herein, which are intended as singleillustrations of individual aspects of the present technology. Manymodifications and variations of this present technology can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. Functionally equivalent methods within thescope of the present technology, in addition to those enumerated herein,will be apparent to those skilled in the art from the foregoingdescriptions. Such modifications and variations are intended to fallwithin the scope of the appended claims. It is to be understood thatthis present technology is not limited to particular methods, reagents,compounds, compositions, labeled compounds or biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular aspects only,and is not intended to be limiting. Thus, it is intended that thespecification be considered as exemplary only with the breadth, scopeand spirit of the present technology indicated only by the appendedclaims, definitions therein and any equivalents thereof.

The embodiments, illustratively described herein may suitably bepracticed in the absence of any element or elements, limitation orlimitations, not specifically disclosed herein. Thus, for example, theterms “comprising,” “including,” “containing,” etc. shall be readexpansively and without limitation. Additionally, the terms andexpressions employed herein have been used as terms of description andnot of limitation, and there is no intention in the use of such termsand expressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the claimed technology.Additionally, the phrase “consisting essentially of” will be understoodto include those elements specifically recited and those additionalelements that do not materially affect the basic and novelcharacteristics of the claimed technology. The phrase “consisting of”excludes any element not specified.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group. Each of the narrowerspecies and subgeneric groupings falling within the generic disclosurealso form part of the invention. This includes the generic descriptionof the invention with a proviso or negative limitation removing anysubject matter from the genus, regardless of whether or not the excisedmaterial is specifically recited herein.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the like,include the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember.

All publications, patent applications, issued patents, and otherdocuments (for example, journals, articles and/or textbooks) referred toin this specification are herein incorporated by reference as if eachindividual publication, patent application, issued patent, or otherdocument was specifically and individually indicated to be incorporatedby reference in its entirety. Definitions that are contained in textincorporated by reference are excluded to the extent that theycontradict definitions in this disclosure.

The present technology may include, but is not limited to, the featuresand combinations of features recited in the following letteredparagraphs, it being understood that the following paragraphs should notbe interpreted as limiting the scope of the claims as appended hereto ormandating that all such features must necessarily be included in suchclaims:

-   A. A compound of Formula I

or a pharmaceutically acceptable salt thereof, wherein

-   -   Z¹ is H or —X¹—W²    -   Z² is OH or NH—W³;    -   Z³ is H or W⁷,    -   α is 0 or 1;    -   X¹ is O, NH, or S;    -   W² and W³ are each independently H, alkyl, cycloalkyl, alkenyl,        cycloalkenyl, alkynyl, aryl, heterocyclyl, heteroaryl,        —CH₂CH₂—(OCH₂CH₂)_(w)—R′ where w is 1, 2, 3, 4, 5, 6, 7, 8, 9,        or 10, or —CH₂CH₂—(OCH₂CH₂)_(x)—OR′ where x is 1, 2, 3, 4, 5, 6,        7, 8, 9, or 10, each of which may optionally be substituted with        one or more of halo, —N₃, —OR′, —CH₂CH₂—(OCH₂CH₂)_(y)—R′ where y        is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —CH₂CH₂—(OCH₂CH₂)_(z)—OR′        where z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —SR′, —OC(O)R′,        —C(O)OR′, —C(S)OR′, —S(O)R′, —SO₂R′, —SO₂(OR′), —SO₂NR′₂,        —P(O)(OR′)₂, —P(O)R′(OR′), —P(O)R′₂, —CN, —OCN, —SCN, —NCO,        —NCS, —NR′—NH₂, —N═C═N—R′, —SO₂Cl, —C(O)Cl, or an epoxide group,    -   W⁵ and W⁷ are each independently OH, NH₂, SH, alkyl, cycloalkyl,        alkenyl, cycloalkenyl, alkynyl, aryl, heterocyclyl, heteroaryl,        —CH₂CH₂—(OCH₂CH₂)_(w)—R′ where w is 1, 2, 3, 4, 5, 6, 7, 8, 9,        or 10, or —CH₂CH₂—(OCH₂CH₂)_(x)—OR′ where x is 1, 2, 3, 4, 5, 6,        7, 8, 9, or 10, each of which may optionally be substituted with        one or more of halo, —N₃, —OR′, —CH₂CH₂—(OCH₂CH₂)y_(x)-R′ where        y is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —CH₂CH₂—(OCH₂CH₂)_(z)—OR′        where z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —SR′, —OC(O)R′,        —C(O)OR′, —C(S)OR′, —S(O)R′, —SO₂R′, —SO₂(OR′), —SO₂NR′₂,        —P(O)(OR′)₂, —P(O)R′(OR′), —P(O)R′₂, —CN, —OCN, —SCN, —NCO,        —NCS, —NR′—NH₂, —N═C═N—R′, —SO₂Cl, —C(O)Cl, or an epoxide group;        and    -   R′ is independently at each occurrence H, halo, —N₃, C₁-C₆        alkyl, C₃-C₆ cycloalkyl, C₂-C₆ alkenyl, C₅-C₈cycloalkenyl, C₂-C₆        alkynyl, C₈-C₁₀ cycloalkynyl, C₅-C₆ aryl, heterocyclyl, or        heteroaryl.

-   B. The compound of Paragraph A, wherein the compound is of Formula    III

or a pharmaceutically acceptable salt thereof.

-   C. The compound of Paragraph A or Paragraph B, wherein the compound    is

or pharmaceutically acceptable salt thereof

-   D. The compound of Paragraph A, wherein the compound of Formula I is    of Formula VI

or a pharmaceutically acceptable salt thereof.

-   E. The compound of Paragraph A, wherein the compound of Formula I is    of Formula IX

or a pharmaceutically acceptable salt thereof.

-   F. The compound of Paragraph A, wherein the compound of Formula I is    of Formula XII

or a pharmaceutically acceptable salt thereof.

-   G. A compound of Formula IA

or a pharmaceutically acceptable salt thereof, wherein

-   -   M¹ is an alpha-emitting radionuclide;    -   Z¹ is H or —X¹—W²;    -   Z² is OH or NH—W³;    -   Z³ is H or W⁷;    -   α is 0 or 1;    -   X¹ is O, NH, or S;    -   W² and W³ are each independently H, alkyl, cycloalkyl, alkenyl,        cycloalkenyl, alkynyl, aryl, heterocyclyl, heteroaryl,        —CH₂CH₂—(OCH₂CH₂)_(w)—R′ where w is 1, 2, 3, 4, 5, 6, 7, 8, 9,        or 10, or —CH₂CH₂—(OCH₂CH₂)_(x)—OR′ where x is 1, 2, 3, 4, 5, 6,        7, 8, 9, or 10, each of which may optionally be substituted with        one or more of halo, —N₃, —OR′, —CH₂CH₂—(OCH₂CH₂)_(y)—R′ where y        is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —CH₂CH₂—(OCH₂CH₂)_(z)—OR′        where z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —SR′, —OC(O)R′,        —C(O)OR′, —C(S)OR′, —S(O)R′, —SO₂R′, —SO₂(OR′), —SO₂NR′₂,        —P(O)(OR′)₂, —P(O)R′(OR′), —P(O)R′₂, —CN, —OCN, —SCN, —NCO,        —NCS, —NR′—NH₂, —N═C═N—R′, —SO₂Cl, —C(O)Cl, or an epoxide group;    -   W⁵ and W⁷ are each independently OH, NH₂, SH, alkyl, cycloalkyl,        alkenyl, cycloalkenyl, alkynyl, aryl, heterocyclyl, heteroaryl,        —CH₂CH₂—(OCH₂CH₂)_(w)—R′ where w is 1, 2, 3, 4, 5, 6, 7, 8, 9,        or 10, or —CH₂CH₂—(OCH₂CH₂)_(x)—OR′ where x is 1, 2, 3, 4, 5, 6,        7, 8, 9, or 10, each of which may optionally be substituted with        one or more of halo, —N₃, —OR′, —CH₂CH₂—(OCH₂CH₂)y_(x)-R′ where        y is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —CH₂CH₂—(OCH₂CH₂)_(z)—OR′        where z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —SR′, —OC(O)R′,        —C(O)OR′, —C(S)OR′, —S(O)R′, —SO₂R′, —SO₂(OR′), —SO₂NR′₂,        —P(O)(OR′)₂, —P(O)R′(OR′), —P(O)R′₂, —CN, —OCN, —SCN, —NCO,        —NCS, —NR′—NH₂, —N═C═N—R′, —SO₂Cl, —C(O)Cl, or an epoxide group;        and    -   R′ is independently at each occurrence H, halo, —N₃, C₁-C₆        alkyl, C₃-C₆ cycloalkyl, C₂-C₆ alkenyl, C₅-C₈ cycloalkenyl,        C₂-C₆ alkynyl, C₈-C₁₀ cycloalkynyl, C₅-C₆ aryl, heterocyclyl, or        heteroaryl.

-   H. The compound of Paragraph G, wherein M¹ is actinium-225    (²²⁵Ac³⁺), radium-223 (²³³Ra²⁺), bismuth-213 (²¹³Bi³⁺), lead-212    (²¹²Pb²⁺ and/or ²¹²Pb⁴⁺), terbium-149 (¹⁴⁹1Tb³⁺), fermium-255    (²⁵⁵Fm³⁺), thorium-227 (²²⁷Th⁴⁺), thorium-226 (²²⁶Th⁴⁺),    astatine-211 (²¹¹At⁺), astatine-217 (²¹⁷At⁺), or uranium-230.

-   I. The compound of Paragraph G or Paragraph H, wherein the compound    of Formula I is of Formula IV

or a pharmaceutically acceptable salt thereof, wherein M² is analpha-emitting radionuclide.

-   J. The compound of Paragraph I, wherein M² is actinium-225    (²²⁵Ac³⁺), radium-223 (²²³Ra²⁺), bismuth-213 (²¹³Bi³), lead-212    (²¹²Pb²⁺ and/or ²¹²Pb⁴⁺), terbium-149 (¹⁴⁹Tb³⁺), fermium-255    (²⁵⁵Fm³⁺), thorium-227 (²²⁷Th⁴⁺), thorium-226 (²²⁵Th⁴⁺),    astatine-211 (²¹¹At⁺), astatine-217 (²¹⁴At⁺), or uranium-230.-   K. The compound of Paragraph I, wherein the compound is

or a pharmaceutically acceptable salt thereof.

-   L. The compound of Paragraph K, wherein M² is actinium-225    (²²⁵Ac³⁺), radium-223 (²³³Ra²⁺), bismuth-213 (²³Bi³), lead-212    (²¹²Pb²⁺ and/or ²¹²Pb⁴⁺), terbium-149 (¹⁴⁹Tb³⁺), fermium-255    (²⁵⁵Fm³⁺), thorium-227 (²²⁷Th⁴⁺), thorium-226 (²²⁶Th⁴⁺),    astatine-211 (²¹¹At⁺), astatine-217 (²¹⁷At⁺), or uranium-230.-   M. The compound of Paragraph G or Paragraph H, wherein the compound    of Formula IA is of Formula VIII

or a pharmaceutically acceptable salt thereof, wherein M³ is analpha-emitting radionuclide.

-   N. The compound of Paragraph M, wherein M³ is actinium-225    (²²⁵Ac³⁺), radium-223 (²³³Ra²⁺), bismuth-213 (²¹³Bi³⁺), lead-212    (²¹²Pb²⁺ and/or ²¹²Pb⁴⁺), terbium-149 (¹⁴⁹T³⁺), fermium-255    (²⁵⁵Fm³⁺), thorium-227 (²²⁷Th⁴⁺), thorium-226 (²²⁶Th⁴⁺),    astatine-211 (²¹¹At⁺), astatine-217 (²¹⁷At⁺), or uranium-230.-   O. The compound of Paragraph G or Paragraph H, wherein the compound    of Formula IA is of Formula X

or a pharmaceutically acceptable salt thereof, wherein M⁴ is analpha-emitting radionuclide.

-   P. The compound of Paragraph O, wherein M⁴ is actinium-225    (²²⁵Ac³⁺), radium-223 (²³³Ra²⁺), bismuth-213 (²¹³Bi³⁺), lead-212    (²¹²Pb²⁺ and/or ²¹²Pb⁴⁺), terbium-149 (¹⁴⁹Tb³⁺), fermium-255    (²⁵⁵Fm³⁺), thorium-227 (²²⁷Th⁴⁺), thorium-226 (²²⁶Th⁴⁺),    astatine-211 (²¹¹At⁺), astatine-217 (²¹⁷At⁺), or uranium-230.-   Q. The compound of Paragraph G or Paragraph H, wherein the compound    of Formula IA is of Formula XIII

or a pharmaceutically acceptable salt thereof, wherein M⁵ is analpha-emitting radionuclide.

-   R. The compound of Paragraph Q, wherein M⁵ is actinium-225    (²²⁵Ac³⁺), radium-223 (²³³Ra²⁺), bismuth-213 (²¹³Bi⁺), lead-212    (²¹²Pb²⁺ and/or ²¹²Pb⁴⁺), terbium-149 (¹⁴⁹Tb³⁺), fermium-255    (²²⁵Fm³⁺), thorium-227 (²²⁷Th⁴⁺), thorium-226 (²²⁶Th⁴⁺),    astatine-211 (²¹¹At⁺), astatine-217 (²¹⁷At⁺), or uranium-230.-   S. A targeting compound of Formula II

or a pharmaceutically acceptable salt thereof, wherein

-   -   M¹ is an alpha-emitting radionuclide;    -   Z¹ is H or -L³-R²²;    -   Z² is OH or NH-L⁴-R²⁴;    -   Z³ is H or -L⁶-R²⁸    -   α is 0 or 1;    -   X¹ is O, NH, or S;    -   L³, L⁴, L⁵, and L⁶ are independently at each occurrence a bond        or a linker group; and    -   R²², R²⁴, R²⁶, and R²⁸ each independently comprises an antibody,        antibody fragment (e.g., an antigen-binding fragment), a binding        moiety, a binding peptide, a binding polypeptide (such as a        selective targeting oligopeptide containing up to 50 amino        acids), a binding protein, an enzyme, a nucleobase-containing        moiety (such as an oligonucleotide, DNA or RNA vector, or        aptamer), or a lectin.

-   T. The targeting compound of Paragraph S, wherein M¹ is actinium-225    (²²⁵Ac³⁺), radium-223 (²³³Ra²⁺), bismuth-213 (²¹³Bi³⁺), lead-212    (²¹²Pb²⁺ and/or ²¹²Pb⁴⁺), terbium-149 (¹⁴⁹Tb³⁺), fermium-255    (Z⁵Fm³⁺), thorium-227 (²²⁷Th⁴⁺), thorium-226 (²²⁶Th⁴⁺), astatine-211    (²¹¹At⁺), astatine-217 (²¹⁷At⁺), or uranium-230.

-   U. The targeting compound of Paragraph S or Paragraph T, wherein    R²², R²⁴, R²⁶, and R²⁸ each independently comprise belimumab,    Mogamulizumab, Blinatumomab, Ibritumomab tiuxetan, Obinutuzumab,    Ofatumumab, Rituximab, Inotuzumab ozogamicin, Moxetumomab pasudotox,    Brentuximab vedotin, Daratumumab, Ipilimumab, Cetuximab,    Necitumumab, Panitumumab, Dinutuximab, Pertuzumab, Trastuzumab,    Trastuzumab emtansine, Siltuximab, Cemiplimab, Nivolumab,    Pembrolizumab, Olaratumab, Atezolizumab, Avelumab, Durvalumab,    Capromab pendetide, Elotuzumab, Denosumab, Ziv-aflibercept,    Bevacizumab, Ramucirumab, Tositumomab, Gemtuzumab ozogamicin,    Alemtuzumab, Cixutumumab, Girentuximab, Nimotuzumab, Catumaxomab,    Etaracizumab, an antigen-binding fragment of any thereof, a prostate    specific membrane antigen (“PSMA”) binding peptide, a somatostatin    receptor agonist, a bombesin receptor agonist, a seprase binding    compound, or a binding fragment of any thereof.

-   V. The targeting compound of any one of Paragraphs S-U, wherein the    targeting compound of Formula II is of Formula V

or a pharmaceutically acceptable salt thereof, wherein M² is analpha-emitting radionuclide.

-   W. The targeting compound of Paragraph V, wherein M² is actinium-225    (²²⁵Ac³⁺), radium-223 (²³³Ra²⁺), bismuth-213 (²¹³Bi³⁺), lead-212    (²¹²Pb²⁺ and/or ²¹²Pb⁴⁺), terbium-149 (¹⁴⁹Tb³⁺), fermium-255    (²⁵⁵Fm³⁺), thorium-227 (²²⁷Th⁴⁺), thorium-226 (²²⁶Th⁴⁺),    astatine-211 (²²⁵At⁺), astatine-217 (²¹⁷At⁺), or uranium-230.-   X. The targeting compound of any one of Paragraphs S-U, wherein the    targeting compound of Formula II is of Formula VIII

or a pharmaceutically acceptable salt thereof, wherein M¹ is analpha-emitting radionuclide.

-   Y. The targeting compound of Paragraph X, wherein M¹ is actinium-225    (²²⁵Ac³⁺), radium-223 (²²³Ra²⁺), bismuth-213 (²¹³Bi³⁺), lead-212    (²¹²Pb²⁺ and/or ²¹²Pb⁴⁺), terbium-149 (¹⁴⁹Tb³⁺), fermium-255    (²⁵⁵Fm³⁺), thorium-227 (²²⁷Th⁴⁺), thorium-226 (²²⁶Th⁴⁺),    astatine-211 (²¹¹At⁺), astatine-217 (²¹⁷At⁺), or uranium-230.-   Z. The targeting compound of any one of Paragraphs S-U, wherein the    targeting compound of Formula II is of Formula XI

or a pharmaceutically acceptable salt thereof, wherein M⁴ analpha-emitting radionuclide.

-   AA. The targeting compound of Paragraph Z, wherein M⁴ is    actinium-225 (²²⁵Ac³⁺), radium-223 (²³³Ra²⁺), bismuth-213 (²¹³Bi³⁺),    lead-212 (²¹²Pb²⁺ and/or ²¹²Pb⁴⁺), terbium-149 (⁴⁹Tb³⁺), fermium-255    (²⁵⁵Fm³⁺), thorium-227 (²²⁷Th⁴⁺), thorium-226 (²²⁶Th⁴⁺),    astatine-211 (²¹¹At⁺), astatine-217 (²¹⁷At⁺), or uranium-230.-   AB. The targeting compound of any one of Paragraphs S-U, wherein the    targeting compound of Formula II is of Formula XIV

or a pharmaceutically acceptable salt thereof, wherein M⁵ is analpha-emitting radionuclide.

-   AC. The targeting compound of Paragraph AB, wherein M⁵ is    actinium-225 (²²⁵Ac³⁺), radium-223 (²²³Ra²⁺), bismuth-213 (²¹³Bi³⁺),    lead-212 (²¹²Pb²⁺ and/or ²¹²Pb⁴⁺), terbium-149 (¹⁴⁹Tb³⁺) fermium-255    (²⁵⁵Fm³⁺), thorium-227 (²²⁷Th⁴⁺), thorium-226 (²²⁶Th⁴⁺),    astatine-211 (²¹¹At⁺), astatine-217 (²¹⁷At⁺), or uranium-230.-   AD. A modified antibody, modified antibody fragment, or modified    binding peptide comprising a linkage arising from conjugation of a    compound of Formula I

or pharmaceutically acceptable salt thereof, with an antibody, antibodyfragment, or binding peptide, wherein

-   -   Z¹ is H or —X¹—W²;    -   Z² is OH or NH—W³;    -   Z³ is H or W⁷;    -   α is 0 or 1;    -   X¹ is O, NH, or S;    -   W² and W³ are each independently H, alkyl, cycloalkyl, alkenyl,        cycloalkenyl, alkynyl, aryl, heterocyclyl, heteroaryl,        —CH₂CH₂—(OCH₂CH₂)_(w)—R′ where w is 1, 2, 3, 4, 5, 6, 7, 8, 9,        or 10, or —CH₂CH₂—(OCH₂CH₂)_(x)—OR′ where x is 1, 2, 3, 4, 5, 6,        7, 8, 9, or 10, each of which may optionally be substituted with        one or more of halo, —N₃, —OR′, —CH₂CH₂—(OCH₂CH₂)_(y)—R′ where y        is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —CH₂CH₂—(OCH₂CH₂)_(z)—OR′        where z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —SR′, —OC(O)R′,        —C(O)OR′, —C(S)OR′, —S(O)R′, —SO₂R′, —SO₂(OR′), —SO₂NR′₂,        —P(O)(OR′)₂, —P(O)R′(OR′), —P(O)R′₂, —CN, —OCN, —SCN, —NCO,        —NCS, —NR′—NH₂, —N═C═N—R′, —SO₂Cl, —C(O)Cl, or an epoxide group,    -   W⁵ and W⁷ are each independently OH, NH₂, SH, alkyl, cycloalkyl,        alkenyl, cycloalkenyl, alkynyl, aryl, heterocyclyl, heteroaryl,        —CH₂CH₂—(OCH₂CH₂)_(w)—R′ where w is 1, 2, 3, 4, 5, 6, 7, 8, 9,        or 10, or —CH₂CH₂—(OCH₂CH₂)_(x)—OR′ where x is 1, 2, 3, 4, 5, 6,        7, 8, 9, or 10, each of which may optionally be substituted with        one or more of halo, —N₃, —OR′, —CH₂CH₂—(OCH₂CH₂)y_(x)-R′ where        y is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —CH₂CH₂—(OCH₂CH₂)_(z)—OR′        where z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —SR′, —OC(O)R′,        —C(O)OR′, —C(S)OR′, —S(O)R′, —SO₂R′, —SO₂(OR′), —SO₂NR′₂,        —P(O)(OR′)₂, —P(O)R′(OR′), —P(O)R′₂, —CN, —OCN, —SCN, —NCO,        —NCS, —NR′—NH₂, —N═C═N—R′, —SO₂Cl, —C(O)Cl, or an epoxide group;        and    -   R′ is independently at each occurrence H, halo, —N₃, C₁-C₆        alkyl, C₃-C₆ cycloalkyl, C₂-C₆ alkenyl, C₅-C₈ cycloalkenyl,        C₂-C₆ alkynyl, C₈-C₁₀ cycloalkynyl, C₅-C₆ aryl, heterocyclyl, or        heteroaryl.

-   AE. The modified antibody, modified antibody fragment, or modified    binding peptide of Paragraph AD, wherein the antibody comprises    belimumab, Mogamulizumab, Blinatumomab, Ibritumomab tiuxetan,    Obinutuzumab, Ofatumumab, Rituximab, Inotuzumab ozogamicin,    Moxetumomab pasudotox, Brentuximab vedotin, Daratumumab, Ipilimumab,    Cetuximab, Necitumumab, Panitumumab, Dinutuximab, Pertuzumab,    Trastuzumab, Trastuzumab emtansine, Siltuximab, Cemiplimab,    Nivolumab, Pembrolizumab, Olaratumab, Atezolizumab, Avelumab,    Durvalumab, Capromab pendetide, Elotuzumab, Denosumab,    Ziv-aflibercept, Bevacizumab, Ramucirumab, Tositumomab, Gemtuzumab    ozogamicin, Alemtuzumab, Cixutumumab, Girentuximab, Nimotuzumab,    Catumaxomab, or Etaracizumab.

-   AF. The modified antibody, modified antibody fragment, or modified    binding peptide of Paragraph AD or Paragraph AE, wherein the    antibody fragment comprises an antigen-binding fragment of    belimumab, Mogamulizumab, Blinatumomab, Ibritumomab tiuxetan,    Obinutuzumab, Ofatumumab, Rituximab, Inotuzumab ozogamicin,    Moxetumomab pasudotox, Brentuximab vedotin, Daratumumab, Ipilimumab,    Cetuximab, Necitumumab, Panitumumab, Dinutuximab, Pertuzumab,    Trastuzumab, Trastuzumab emtansine, Siltuximab, Cemiplimab,    Nivolumab, Pembrolizumab, Olaratumab, Atezolizumab, Avelumab,    Durvalumab, Capromab pendetide, Elotuzumab, Denosumab,    Ziv-aflibercept, Bevacizumab, Ramucirumab, Tositumomab, Gemtuzumab    ozogamicin, Alemtuzumab, Cixutumumab, Girentuximab, Nimotuzumab,    Catumaxomab, or Etaracizumab.

-   AG. The modified antibody, modified antibody fragment, or modified    binding peptide of any one of Paragraphs AD-AF, wherein the binding    peptide comprises a prostate specific membrane antigen (“PSMA”)    binding peptide, a somatostatin receptor agonist, a bombesin    receptor agonist, a seprase binding compound, or a binding fragment    thereof. AH. The modified antibody, modified antibody fragment, or    modified binding peptide of any one of Paragraphs AD-AG, wherein the    compound of Formula I is of Formula III

or a pharmaceutically acceptable salt thereof.

-   AI. The modified antibody, modified antibody fragment, or modified    binding peptide of any one of Paragraphs AD-AH, wherein the linkage    is a thiocynate linkage; wherein the thiocyanate linkage arises from    conjugation of the compound with the antibody, antibody fragment, or    binding peptide; and wherein the compound is

or pharmaceutically acceptable salt thereof.

-   AJ. The modified antibody, modified antibody fragment, or modified    binding peptide of any one of Paragraphs AD-AG, wherein the compound    of Formula I is of Formula VI

or a pharmaceutically acceptable salt thereof.

-   AK. The modified antibody, modified antibody fragment, or modified    binding peptide of any one of Paragraphs AD-AG, wherein the compound    of Formula I is of Formula IX

or a pharmaceutically acceptable salt thereof.

-   AL. The modified antibody, modified antibody fragment, or modified    binding peptide of any one of Paragraphs AD-AG, wherein the compound    of Formula I is of Formula XII

or a pharmaceutically acceptable salt thereof.

-   AM. A modified antibody, modified antibody fragment, or modified    binding peptide comprising a linkage arising from conjugation of a    compound of Formula IA

or a pharmaceutically acceptable salt thereof, with an antibody,antibody fragment, or binding peptide, wherein

-   -   M¹ is an alpha-emitting radionuclide;    -   Z¹ is H or —X¹—W²;    -   Z² is OH or NH—W³;    -   Z³ is H or W⁷;    -   α is 0 or 1;    -   X¹ is O, NH, or S;    -   W² and W³ are each independently H, alkyl, cycloalkyl, alkenyl,        cycloalkenyl, alkynyl, aryl, heterocyclyl, heteroaryl,        —CH₂CH₂—(OCH₂CH₂)_(w)—R′ where w is 1, 2, 3, 4, 5, 6, 7, 8, 9,        or 10, or —CH₂CH₂—(OCH₂CH₂)_(x)—OR′ where x is 1, 2, 3, 4, 5, 6,        7, 8, 9, or 10, each of which may optionally be substituted with        one or more of halo, —N₃, —OR′, —CH₂CH₂—(OCH₂CH₂)_(y)—R′ where y        is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —CH₂CH₂—(OCH₂CH₂)_(z)—OR′        where z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —SR′, —OC(O)R′,        —C(O)OR′, —C(S)OR′, —S(O)R′, —SO₂R′, —SO₂(OR′), —SO₂NR′₂,        —P(O)(OR′)₂, —P(O)R′(OR′), —P(O)R′₂, —CN, —OCN, —SCN, —NCO,        —NCS, —NR′—NH₂, —N═C═N—R′, —SO₂Cl, —C(O)Cl, or an epoxide group;    -   W⁵ and W⁷ are each independently OH, NH₂, SH, alkyl, cycloalkyl,        alkenyl, cycloalkenyl, alkynyl, aryl, heterocyclyl, heteroaryl,        —CH₂CH₂—(OCH₂CH₂)_(w)—R′ where w is 1, 2, 3, 4, 5, 6, 7, 8, 9,        or 10, or —CH₂CH₂—(OCH₂CH₂)_(x)—OR′ where x is 1, 2, 3, 4, 5, 6,        7, 8, 9, or 10, each of which may optionally be substituted with        one or more of halo, —N₃, —OR′, —CH₂CH₂—(OCH₂CH₂)y_(x)-R′ where        y is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —CH₂CH₂—(OCH₂CH₂)_(z)—OR′        where z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —SR′, —OC(O)R′,        —C(O)OR′, —C(S)OR′, —S(O)R′, —SO₂R′, —SO₂(OR′), —SO₂NR′₂,        —P(O)(OR′)₂, —P(O)R′(OR′), —P(O)R′₂, —CN, —OCN, —SCN, —NCO,        —NCS, —NR′—NH₂, —N═C═N—R′, —SO₂Cl, —C(O)Cl, or an epoxide group;        and R′ is independently at each occurrence H, halo, —N₃, C₁-C₆        alkyl, C₃-C₆ cycloalkyl, C₂-C₆ alkenyl, C₅-C₈ cycloalkenyl,        C₂-C₆ alkynyl, C₈-C₁₀ cycloalkynyl, C₅-C₆ aryl, heterocyclyl, or        heteroaryl.

-   AN. The modified antibody, modified antibody fragment, or modified    binding peptide of Paragraph AM, wherein M¹ is actinium-225    (²²⁵Ac³), radium-223 (²²³Ra²⁺), bismuth-213 (²¹³Bi³⁺), lead-212    (²¹²Pb²⁺ and/or ²¹²Pb⁴⁺), terbium-149 (¹⁴⁹Tb³⁺), fermium-255    (²⁵⁵Fm³⁺), thorium-227 (²²¹Th⁴⁺), thorium-226 (²²⁶Th⁴⁺),    astatine-211 (²¹¹At⁺), astatine-217 (²¹⁷At⁺), or uranium-230.

-   AO. The modified antibody, modified antibody fragment, or modified    binding peptide of Paragraph AM or Paragraph AN, wherein the    antibody comprises belimumab, Mogamulizumab, Blinatumomab,    Ibritumomab tiuxetan, Obinutuzumab, Ofatumumab, Rituximab,    Inotuzumab ozogamicin, Moxetumomab pasudotox, Brentuximab vedotin,    Daratumumab, Ipilimumab, Cetuximab, Necitumumab, Panitumumab,    Dinutuximab, Pertuzumab, Trastuzumab, Trastuzumab emtansine,    Siltuximab, Cemiplimab, Nivolumab, Pembrolizumab, Olaratumab,    Atezolizumab, Avelumab, Durvalumab, Capromab pendetide, Elotuzumab,    Denosumab, Ziv-aflibercept, Bevacizumab, Ramucirumab, Tositumomab,    Gemtuzumab ozogamicin, Alemtuzumab, Cixutumumab, Girentuximab,    Nimotuzumab, Catumaxomab, or Etaracizumab.

-   AP. The modified antibody, modified antibody fragment, or modified    binding peptide of any one of Paragraphs AM-AO, wherein the antibody    fragment comprises an antigen-binding fragment of belimumab,    Mogamulizumab, Blinatumomab, Ibritumomab tiuxetan, Obinutuzumab,    Ofatumumab, Rituximab, Inotuzumab ozogamicin, Moxetumomab pasudotox,    Brentuximab vedotin, Daratumumab, Ipilimumab, Cetuximab,    Necitumumab, Panitumumab, Dinutuximab, Pertuzumab, Trastuzumab,    Trastuzumab emtansine, Siltuximab, Cemiplimab, Nivolumab,    Pembrolizumab, Olaratumab, Atezolizumab, Avelumab, Durvalumab,    Capromab pendetide, Elotuzumab, Denosumab, Ziv-aflibercept,    Bevacizumab, Ramucirumab, Tositumomab, Gemtuzumab ozogamicin,    Alemtuzumab, Cixutumumab, Girentuximab, Nimotuzumab, Catumaxomab, or    Etaracizumab.

-   AQ. The modified antibody, modified antibody fragment, or modified    binding peptide of any one of Paragraphs AM-AP, wherein the binding    peptide comprises a prostate specific membrane antigen (“PSMA”)    binding peptide, a somatostatin receptor agonist, a bombesin    receptor agonist, a seprase binding compound, or a binding fragment    thereof.

-   AR. The modified antibody, modified antibody fragment, or modified    binding peptide of any one of Paragraphs AM-AQ, wherein the compound    of Formula I is of Formula IV

or a pharmaceutically acceptable salt thereof, wherein M² is analpha-emitting radionuclide.

-   AS. The modified antibody, modified antibody fragment, or modified    binding peptide of Paragraph AR, wherein M² is actinium-225    (²²⁵Ac³⁺), radium-223 (²³³Ra²⁺), bismuth-213 (²¹³Bi³⁺), lead-212    (²¹²Pb²⁺ and/or ²¹²Pb⁴⁺), terbium-149 (¹⁴⁹Tb³⁺), fermium-255    (²⁵⁵Fm³⁺), thorium-227 (²²⁷Th⁴⁺), thorium-226 (²²⁶Th⁴⁺),    astatine-211 (²¹¹At⁺), astatine-217 (²¹⁷AC), or uranium-230.-   AT. The modified antibody, modified antibody fragment, or modified    binding peptide of Paragraph AR, wherein the linkage is a thiocynate    linkage; wherein the thiocyanate linkage arises from conjugation of    the compound with the antibody, antibody fragment, or binding    peptide; and wherein the compound is

or a pharmaceutically acceptable salt thereof.

-   AU. The modified antibody, modified antibody fragment, or modified    binding peptide of Paragraph AT, wherein M² is actinium-225    (²⁷⁵Ac³⁺), radium-223 (²²³Ra²⁺), bismuth-213 (²¹³Bi³⁺), lead-212    (²¹²Pb²⁺ and/or ²¹²Pb⁴⁺), terbium-149 (¹⁴⁹Tb³⁺), fermium-255    (²⁵⁵Fm³⁺), thorium-227 (²²⁷Th⁴⁺), thorium-226 (²²⁶Th⁴⁺),    astatine-211 (²¹¹At⁺), astatine-217 (²¹⁷At⁺), or uranium-230.-   AV. The modified antibody, modified antibody fragment, or modified    binding peptide of any one of Paragraphs AM-AQ, wherein the compound    of Formula IA is of Formula VIII

or a pharmaceutically acceptable salt thereof, wherein M³ is analpha-emitting radionuclide.

-   AW. The modified antibody, modified antibody fragment, or modified    binding peptide of Paragraph AV, wherein M³ is actinium-225    (²²⁵Ac³⁺), radium-223 (²²³Ra²⁺), bismuth-213 (²¹³Bi³⁺), lead-212    (²¹²Pb²˜ and/or ²¹²Pb⁴⁺), terbium-149 (¹⁴⁹Tb³⁺), fermium-255    (²⁵⁵Fm³⁺), thorium-227 (²²⁷Th⁴⁺), thorium-226 (²²⁶Th⁴⁺),    astatine-211 (²¹¹At⁺), astatine-217 (²¹⁷At⁺), or uranium-230.-   AX. The modified antibody, modified antibody fragment, or modified    binding peptide of any one of Paragraphs AM-AQ, wherein the compound    of Formula IA is of Formula X

or a pharmaceutically acceptable salt thereof, wherein M⁴ is analpha-emitting radionuclide.

-   AY. The modified antibody, modified antibody fragment, or modified    binding peptide of Paragraph AX, wherein M⁴ is actinium-225    (²²⁵Ac³⁺), radium-223 (²²³Ra²⁺), bismuth-213 (²¹³Bi³⁺), lead-212    (²¹²Pb²⁺ and/or ²¹²Pb⁴⁺), terbium-149 (¹⁴⁹Tb³⁺), fermium-255    (²⁵⁵Fm³⁺), thorium-227 (²²⁷Th⁴⁺), thorium-226 (²²⁶Th⁴⁺),    astatine-211 (²¹¹At⁺), astatine-217 (²¹⁷At⁺), or uranium-230.-   AZ. The modified antibody, modified antibody fragment, or modified    binding peptide of any one of Paragraphs AM-AQ, wherein the compound    of Formula IA is of Formula XIII

or a pharmaceutically acceptable salt thereof, wherein M⁵ is analpha-emitting radionuclide.

-   BA. The modified antibody, modified antibody fragment, or modified    binding peptide of Paragraph AZ, wherein M⁵ is actinium-225    (²⁷⁵Ac³⁺), radium-223 (²²³Ra²⁺), bismuth-213 (²¹³Bi³⁺), lead-212    (²¹²Pb²⁺ and/or ²¹²Pb⁴⁺), terbium-149 (¹⁴⁹Tb³⁺), fermium-255    (²⁵⁵Fm³⁺), thorium-227 (²²⁷Th⁴⁺), thorium-226 (²²⁶Th⁴⁺),    astatine-211 (²¹¹At⁺), astatine-217 (²¹⁷At⁺), or uranium-230.-   BB. A composition comprising a pharmaceutically acceptable carrier    and a compound of any one of Paragraphs A-R.-   BC. A composition comprising a pharmaceutically acceptable carrier    and a targeting compound of any one of Paragraphs S-AC or comprising    a pharmaceutically acceptable carrier and a modified antibody,    modified antibody fragment, or modified binding peptide of any one    of Paragraphs AD-BA.-   BD. A pharmaceutical composition useful in targeted radiotherapy of    cancer and/or mammalian tissue overexpressing prostate specific    membrane antigen (“PSMA”) in a subject, wherein the pharmaceutical    composition comprises a pharmaceutically acceptable carrier and a    compound of any one of Paragraphs S-AC or a modified antibody,    modified antibody fragment, or modified binding peptide of any one    of Paragraphs AD-BA.-   BE. The pharmaceutical composition of Paragraph BD, wherein the    pharmaceutical composition comprises an effective amount for    treating the cancer and/or mammalian tissue overexpressing PSMA of    the compound or an effective amount for treating the cancer and/or    mammalian tissue overexpressing PSMA of the modified antibody,    modified antibody fragment, or modified binding peptide.-   BF. The pharmaceutical composition of Paragraph BD or Paragraph BE,    where the subject suffers from a mammalian tissue expressing a    somatostatin receptor, a bombesin receptor, seprase, or a    combination of any two or more thereof, and/or mammalian tissue    overexpressing PSMA.-   BG. The pharmaceutical composition of any one of Paragraphs BD-BF,    wherein the subject suffers from one or more of a growth hormone    producing tumor, a neuroendocrine tumor, a pituitary tumor, a    vasoactive intestinal peptide-secreting tumor, a small cell    carcinoma of the lung, gastric cancer tissue, pancreatic cancer    tissue, a neuroblastoma,-   BH. The pharmaceutical composition of any one of Paragraphs BD-BG,    wherein the subject suffers from one or more of a glioma, a breast    cancer, an adrenal cortical cancer, a cervical carcinoma, a vulvar    carcinoma, an endometrial carcinoma, a primary ovarian carcinoma, a    metastatic ovarian carcinoma, a non-small cell lung cancer, a small    cell lung cancer, a bladder cancer, a colon cancer, a primary    gastric adenocarcinoma, a primary colorectal adenocarcinoma, a renal    cell carcinoma, and a prostate cancer.-   BI. The pharmaceutical composition of any one of Paragraphs BD-BH,    wherein the pharmaceutical composition is formulated for intravenous    administration, optionally comprising sterilized water, Ringer's    solution, or an isotonic aqueous saline solution.-   BJ. The pharmaceutical composition of any one of Paragraphs BD-BI,    wherein the effective amount of the compound is from about 0.01 μg    to about 10 mg of the compound per gram of the pharmaceutical    composition.-   BK. The pharmaceutical composition of any one of Paragraphs BD-BJ,    wherein the pharmaceutical composition is provided in an injectable    dosage form.-   BL. A method of treating a subject, wherein the method comprises    administering a targeting compound of any one of Paragraphs S-AC to    the subject or administering a modified antibody, modified antibody    fragment, or modified binding peptide of any one of Paragraphs    AD-BA.-   BM. The method of Paragraph BL, wherein the subject suffers from    cancer and/or mammalian tissue overexpressing prostate specific    membrane antigen (“PSMA”)-   BN. The method of Paragraph BM, wherein the method comprises    administering an effective amount for treating the cancer and/or    mammalian tissue overexpressing PSMA of the compound or an effective    amount for treating the cancer and/or mammalian tissue    overexpressing PSMA of the modified antibody, modified antibody    fragment, or modified binding peptide-   BO. The method of any one of Paragraphs BL-BN, wherein the subject    suffers from a mammalian tissue expressing a somatostatin receptor,    a bombesin receptor, seprase, or a combination of any two or more    thereof and/or mammalian tissue overexpressing prostate specific    membrane antigen (“PSMA”), when administered to a subject.-   BP. The method of any one of Paragraphs BL-BO, wherein the mammalian    tissue comprises one or more of a growth hormone producing tumor, a    neuroendocrine tumor, a pituitary tumor, a vasoactive intestinal    peptide-secreting tumor, a small cell carcinoma of the lung, gastric    cancer tissue, pancreatic cancer tissue, a neuroblastoma, and a    metastatic cancer.-   BQ. The method of any one of Paragraphs BL-BP, wherein the subject    suffers from one or more of a glioma, a breast cancer, an adrenal    cortical cancer, a cervical carcinoma, a vulvar carcinoma, an    endometrial carcinoma, a primary ovarian carcinoma, a metastatic    ovarian carcinoma, a non-small cell lung cancer, a small cell lung    cancer, a bladder cancer, a colon cancer, a primary gastric    adenocarcinoma, a primary colorectal adenocarcinoma, a renal cell    carcinoma, and a prostate cancer.-   BR. The method of any one of Paragraphs BL-BQ, wherein the    administering comprises parenteral administration.-   BS. The method of any one of Paragraphs BL-BR, wherein the    administering comprises intravenous administration.-   BT. The method of any one of Paragraphs BL-BS, wherein the effective    amount is from about 0.1 μg to about 50 μg per kilogram of subject    mass.-   BU. A compound comprising a first domain having a blood-protein    binding moiety with low specific affinity for the blood-protein, a    second domain having a tumor targeting moiety with high affinity for    a tumor antigen, and a third domain having a chelator. BV. The    compound of Paragraph BU, wherein the tumor antigen is PSMA,    bombesin, somatostatin receptor, or seprase.-   BW. The compound of Paragraph BU or Paragraph BV, wherein the blood    protein binding moiety has specific affinity for albumin of about    0.5-50×10⁻⁶M, and the tumor targeting moiety has specific affinity    for the tumor antigen of about 0.5-50×10⁻⁹M.-   BX. A compound represented by the following structure

or a pharmaceutically acceptable salt thereof.

-   BY. A composition comprising the compound of Paragraph BX chelating    ²¹³Bi³⁺, ²¹¹At⁺, ²²⁵Ac³⁺, ¹⁵²Dy³⁺, ²¹²Bi³⁺, ²¹¹Bi³⁺, ²¹⁷At⁺,    ²²⁷Th⁴⁺, ²²⁶Th⁴⁺, ²³³Ra²⁺, ²¹²Pb²⁺, or ²¹²Pb⁴⁺.-   BZ. A method of treating a subject, wherein the method comprises    administering a composition of Paragraph BY to the subject.-   CA. The method of Paragraph BZ, wherein the subject suffers from    cancer and/or mammalian tissue overexpressing prostate specific    membrane antigen (“PSMA”)-   CB. The method of Paragraph CA, wherein the method comprises    administering an effective amount for treating the cancer and/or    mammalian tissue overexpressing PSMA of the composition.-   CC. The method of any one of Paragraphs BZ-CB, wherein the subject    suffers from a mammalian tissue overexpressing prostate specific    membrane antigen (“PSMA”).-   CD. The method of any one of Paragraphs BZ-CC, wherein the mammalian    tissue comprises one or more of a growth hormone producing tumor, a    neuroendocrine tumor, a pituitary tumor, a vasoactive intestinal    peptide-secreting tumor, a small cell carcinoma of the lung, gastric    cancer tissue, pancreatic cancer tissue, a neuroblastoma, and a    metastatic cancer.-   CE. The method of any one of Paragraphs BZ-CD, wherein the subject    suffers from one or more of a glioma, a breast cancer, an adrenal    cortical cancer, a cervical carcinoma, a vulvar carcinoma, an    endometrial carcinoma, a primary ovarian carcinoma, a metastatic    ovarian carcinoma, a non-small cell lung cancer, a small cell lung    cancer, a bladder cancer, a colon cancer, a primary gastric    adenocarcinoma, a primary colorectal adenocarcinoma, a renal cell    carcinoma, and a prostate cancer.-   CF. The method of any one of Paragraphs BZ-CE, wherein the    administering comprises parenteral administration.-   CG. The method of any one of Paragraphs BZ-CF, wherein the    administering comprises intravenous administration.-   CH. The method of any one of Paragraphs BZ-CG, wherein the effective    amount is from about 0.1 μg to about 50 μg per kilogram of subject    mass.

Other embodiments are set forth in the following claims, along with thefull scope of equivalents to which such claims are entitled.

What is claimed is:
 1. A compound of Formula I

or a pharmaceutically acceptable salt thereof, wherein Z¹ is H or—X¹—W²; Z² is OH or NH—W³; Z³ is H or W⁷; where at least one of Z¹ andZ³ is not H or Z² is not OH, α is 0 or 1; X¹ is O, NH, or S; W² and W³are each independently H, alkyl, cycloalkyl, alkenyl, cycloalkenyl,alkynyl, aryl, heterocyclyl, heteroaryl, —CH₂CH₂—(OCH₂CH₂)_(w)—R′ wherew is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or —CH₂CH₂—(OCH₂CH₂)_(x)—OR′where x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, each of which mayoptionally be substituted with one or more of halo, —N₃, —OR′,—CH₂CH₂—(OCH₂CH₂)_(y)—R′ where y is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10,—CH₂CH₂—(OCH₂CH₂)_(z)—OR′ where z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10,—SR′, —OC(O)R′, —C(O)OR′, —C(S)OR′, —S(O)R′, —SO₂R′, —SO₂(OR′),—SO₂NR′₂, —P(O)(OR′)₂, —P(O)R′(OR′), —P(O)R′₂, —CN, —OCN, —SCN, —NCO,—NCS, —NR′—NH₂, —N═C═N—R′, —SO₂Cl, —C(O)Cl, or an epoxide group; W⁵ andW⁷ are each independently OH, NH₂, SH, alkyl, cycloalkyl, alkenyl,cycloalkenyl, alkynyl, aryl, heterocyclyl, heteroaryl,—CH₂CH₂—(OCH₂CH₂)_(w)—R′ where w is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or—CH₂CH₂—(OCH₂CH₂)_(x)—OR′ where x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10,each of which may optionally be substituted with one or more of halo,—N₃, —OR′, —CH₂CH₂—(OCH₂CH₂)y_(x)-R′ where y is 1, 2, 3, 4, 5, 6, 7, 8,9, or 10, —CH₂CH₂—(OCH₂CH₂)_(z)—OR′ where z is 1, 2, 3, 4, 5, 6, 7, 8,9, or 10, —SR′, —OC(O)R′, —C(O)OR′, —C(S)OR′, —S(O)R′, —SO₂R′,—SO₂(OR′), —SO₂NR′₂, —P(O)(OR′)₂, —P(O)R′(OR′), —P(O)R′₂, —CN, —OCN,—SCN, —NCO, —NCS, —NR′—NH₂, —N═C═N—R′, —SO₂Cl, —C(O)Cl, or an epoxidegroup; and R′ is independently at each occurrence H, halo, —N₃, C₁-C₆alkyl, C₃-C₆ cycloalkyl, C₂-C₆ alkenyl, C₅-C₈cycloalkenyl, C₂-C₆alkynyl, C₈-C₁₀ cycloalkynyl, C₅-C₆ aryl, heterocyclyl, or heteroaryl.2. The compound of claim 1, wherein W² and W³ are each independentlyalkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heterocyclyl,heteroaryl, —CH₂CH₂—(OCH₂CH₂)_(w)—R′ where w is 1, 2, 3, 4, 5, 6, 7, 8,9, or 10, or —CH₂CH₂—(OCH₂CH₂)_(x)—OR′ where x is 1, 2, 3, 4, 5, 6, 7,8, 9, or 10, each of which may optionally be substituted with one ormore of halo, —N₃, —OR′, —CH₂CH₂—(OCH₂CH₂)_(y)—R′ where y is 1, 2, 3, 4,5, 6, 7, 8, 9, or 10, —CH₂CH₂—(OCH₂CH₂)_(z)—OR′ where z is 1, 2, 3, 4,5, 6, 7, 8, 9, or 10, —SR′, —OC(O)R′, —C(O)OR′, —C(S)OR′, —S(O)R′,—SO₂R′, —SO₂(OR′), —SO₂NR′₂, —P(O)(OR′)₂, —P(O)R′(OR′), —P(O)R′₂, —CN,—OCN, —SCN, —NCO, —NCS, —NR′—NH₂, —N═C═N—R′, —SO₂Cl, —C(O)Cl, or anepoxide group.
 3. The compound of claim 1, wherein W² and W³ are eachindependently alkyl, cycloalkyl, alkynyl, aryl, heterocyclyl,heteroaryl, —CH₂CH₂—(OCH₂CH₂)_(w)—R′ where w is 1, 2, 3, 4, 5, 6, 7, 8,9, or 10, or —CH₂CH₂—(OCH₂CH₂), OR′ where x is 1, 2, 3, 4, 5, 6, 7, 8,9, or 10, each of which may optionally be substituted with one or moreof halo, —N₃, —OR′, —CH₂CH₂—(OCH₂CH₂)_(y)—R′ where y is 1, 2, 3, 4, 5,6, 7, 8, 9, or 10, —CH₂CH₂—(OCH₂CH₂)_(z)—OR′ where z is 1, 2, 3, 4, 5,6, 7, 8, 9, or 10, —SR′, —OC(O)R′, —C(O)OR′, —C(S)OR′, —S(O)R′, —SO₂R′,—SO₂(OR′), —SO₂NR′₂, —P(O)(OR′)₂, —P(O)R′(OR′), —P(O)R′₂, —CN, —OCN,—SCN, —NCO, —NCS, —NR′—NH₂, —N═C═N—R′, —SO₂Cl, —C(O)Cl, or an epoxidegroup.
 4. The compound of claim 1, wherein W⁵ and W⁷ are eachindependently OH, NH₂, SH, alkyl, cycloalkyl, alkynyl, aryl,heterocyclyl, heteroaryl, —CH₂CH₂—(OCH₂CH₂)_(w)—R′ where w is 1, 2, 3,4, 5, 6, 7, 8, 9, or 10, or —CH₂CH₂—(OCH₂CH₂)_(x)—OR′ where x is 1, 2,3, 4, 5, 6, 7, 8, 9, or 10, each of which may optionally be substitutedwith one or more of halo, —N₃, —OR′, —CH₂CH₂—(OCH₂CH₂)y_(x)-R′ where yis 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —CH₂CH₂—(OCH₂CH₂)_(z)—OR′ where zis 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, —SR′, —OC(O)R′, —C(O)OR′, —C(S)OR′,—S(O)R′, —SO₂R′, —SO₂(OR′), —SO₂NR′₂, —P(O)(OR′)₂, —P(O)R′(OR′),—P(O)R′₂, —CN, —OCN, —SCN, —NCO, —NCS, —NR′—NH₂, —N═C═N—R′, —SO₂Cl,—C(O)Cl, or an epoxide group.
 5. The compound of claim 1, wherein thecompound is of Formula III

or a pharmaceutically acceptable salt thereof.
 6. The compound of claim5, wherein W² is

where n is independently at each occurrence 1, 2, 3, 4, 5, 6, 7, 8, 9,or
 10. 7. The compound of claim 5, wherein W² is

where n is independently at each occurrence 1, 2, 3, 4, 5, 6, 7, 8, 9,or
 10. 8. The compound of claim 5, wherein W² is

where n is independently at each occurrence 1, 2, 3, 4, 5, 6, 7, 8, 9,or
 10. 9. The compound of claim 1, wherein the compound is

or pharmaceutically acceptable salt thereof.
 10. The compound of claim1, wherein the compound of Formula I is of Formula VI

or a pharmaceutically acceptable salt thereof.
 11. The compound of claim10, wherein W³ is

where n is independently at each occurrence 1, 2, 3, 4, 5, 6, 7, 8, 9,or
 10. 12. The compound of claim 1, wherein the compound of Formula I isof Formula IX

or a pharmaceutically acceptable salt thereof.
 13. The compound of claim12, wherein W⁵ is

where n is independently at each occurrence 1, 2, 3, 4, 5, 6, 7, 8, 9,or
 10. 14. The compound of claim 1, wherein the compound of Formula I isof Formula XII

or a pharmaceutically acceptable salt thereof.
 15. The compound of claim14, wherein W is

where n is independently at each occurrence 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10.